Detection and treatment of breast disease

ABSTRACT

An isolated chemokine is disclosed. The isolated chemokine is expressed preferentially in breast tissue or can be detected in breast milk. It includes from about 100 to about 132 amino acids, has a deduced molecular weight of from about 10 to about 16 kDa, and has a deduced isoionic point of from about pH 10.1 to about pH 10.7. Antibodies and binding portions thereof recognizing the subject chemokine and peptides which include the antigenic portions of the subject chemokines are described. DNA molecules which encode the subject chemokines as well as nucleic acid molecules which, under stringent conditions, hybridize to nucleic acid molecules encoding the subject chemokines or to a complement thereof are also disclosed. The chemokines, peptides, antibodies and binding portions thereof, and nucleic acid molecules can be used to detect and treat breast disease, such as inflammations, infections, mastitis, benign cystitis, benign hyperplasias, cancer and other malignancies as well as other pathological states of the mammary gland.

[0001] This application is a divisional application of U.S. patentapplication Ser. No. 09/146,580, filed Sep. 3, 1998, which claims thebenefit of U.S. Provisional Patent Application Serial No. 60/071,899,filed Jan. 20, 1998, and U.S. Provisional Patent Application Serial No.60/092,155, filed Jul. 9, 1998, which are hereby incorporated byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to the detection and treatment ofbreast disease.

BACKGROUND OF THE INVENTION

[0003] Breast cancer is one of the largest classes of malignant diseasein women. However, breast cancer presents inherent difficulties inregard to the ease with which it is detected and diagnosed. This is incontrast to detection of some other common cancers, including skin andcervical cancers, the latter of which is based on cytomorphologicscreening techniques.

[0004] Early detection of breast cancer represents a compelling goal inoncology. Although techniques such as computerized tomography,mammography, and magnetic resonance imaging have greatly improved tumorsurveillance over the past decade, there still remains a need forserologic and other blood-based assays.

[0005] Serologic assays are easily performed, inexpensive, andanalytically-sensitive and can be serially run over time with relativeease. The essence of breast cancer screening, using tumor markerdetection, is to efficiently identify a group of higher-risk individualsfrom within a large population. Thereafter, confirmatory testing isimplemented to establish a diagnosis of malignancy.

[0006] There are several classifications of tumor markers possible,based upon the structure or biological function of the marker. Tumormarker classifications include tissue specific antigens (e.g., PSA, NSE,PAP, calcitonin, HCG), major histocompatibility complex (“MHC”)antigens, viral antigens (e.g., HTLV-I gag protein), oncogene products(e.g., c-HER-2/Neu), oncofetal markers (e.g., CEA, AFP), hormones (e.g.,thyroid hormones), enzymes (e.g., telomerase, galactosyltransferase),and altered glycoproteins/glycolipids (e.g., polymorphic epithelialmucins). It should be noted that these classification schemes areimprecise and contain redundancies. For example, calcitonin is animportant serological marker for medullary carcinoma of the thyroid andmay be classified not only as a hormone but also as a tissue specificprotein of the thyroid. Likewise, PSA, HCG, thyroid hormones, PAP, andNSE are tissue specific proteins and also exhibit enzymatic or hormonalactivities. Generally, tumor markers providing high clinical utilityreside in the broadly defined tissue specific class. This class of tumormarkers contains enzymes, isoenzymes, hormones, growth factors, andother molecules with biologic activity.

[0007] The importance of a tumor marker's being tissue specific isillustrated by one of the best known tumor antigens, carcinoembryonicantigen (“CEA”). When first discovered, CEA was thought to be specificto cancers of the digestive system. However, CEA has since been detectedin normal adults as well as in patients with benign liver disease, suchas alcoholic hepatitis or biliary obstruction. Because of the overalllack of specificity and sensitivity, there being no threshold differencein CEA levels that serves to separate benign from malignant conditions,CEA cannot be used in a general diagnostic test. Instead, it isprincipally used to monitor a patient's response to treatment.

[0008] To be useful in serologic assays, a tumor marker should be onethat is released into the bloodstream as a circulating marker.Circulating antigens are now known to exist in breast cancer. Breasttissue markers, such as casein (Franchimont et al., Cancer, 39:2806-2812(1977)) and α-lactalbumin (Kleinberg et al., Science 190:276-278 (1975))and purported cancer markers, such as glycosyl transferases (Ip et al.,Cancer Res., 38:723-728 (1978) and Dao et al., J. Natl. Cancer Inst.,65:529-534 (1980)), glycolipids (Kloppel et al., Proc. Natl. Acad. Sci.USA, 74:3011-3013 (1977)), and phospholipids (Skipski et al., Proc. Soc.Exp. Biol. Med., 136:1261-1264 (1971)) have all been used in variousdiagnostic techniques for breast cancer but have not gained widespreadacceptance as breast cancer markers. More recently, circulating humanmammary epithelial antigens have been proposed as specific markers forbreast cancer (Ceriani et al., Proc. Natl. Acad. Sci. USA, 79:5420-5424(1982)). Burchell et al., Int. J. Cancer, 34:763-768 (1984) describesmonoclonal antibodies which detect high molecular weight mucin-likeantigens elevated in patient serum. Hayes, J. Clin. Invest.,75:1671-1678 (1985) also describes a monoclonal antibody that recognizesa high molecular weight mammary epithelial antigen present in elevatedamounts in the plasma of breast cancer patients. See also Papsidero etal., Cancer Res., 44:4653-4657 (1984) and Taylor-Papadimitriou et al.,Int. J. Cancer., 28:17-28 (1981). Other breast tissue specific proteinsor markers include alpha, beta, and kappa caseins, alpha-lactalbumin,lactoferrin, and selected epithelial membrane antigens. These aredescribed in Cohen et al., Cancer, 60:1294-1298 (1987); Bartkova, Eur.J. Cancer Clin. Oncol., 23:1557-1563 (1987); Weir et al., Cancer Detect.Prev., 4:193-204 (1981); de Almeida et al., Breast Cancer Res. Treat.,21:201-210 (1992); Skilton et al., Tumor Biol., 11:20-38 (1990); Earl etal., Cancer Res., 49:6070-6076 (1989); Barry et al., Amer. J. Clin.Path., 82:582-585 (1984); and Watson et al., Cancer Res., 56:860-865(1996). None of these previously described antigens has been used as abasis for a widely accepted breast cancer clinical assay.

[0009] There have also been several attempts to develop improved methodsof breast cancer detection and diagnosis based on oncogene mutations,gene amplification, and loss of heterozygosity in invasive breastcancer. These methods have not gained wide acceptance.

[0010] Despite the use of mammography and the development of some breasttissue specific markers, there still remains a need for simple and rapidmethods for detecting breast cancer. The present invention is directedto meeting this need.

SUMMARY OF THE INVENTION

[0011] One aspect of the present invention relates to an isolatedchemokine that is preferentially expressed in breast tissue or which canbe detected in breast milk. The isolated chemokine includes about fromabout 100 to about 132 amino acids, has a deduced molecular weight offrom about 10 to about 16 kDa, and has a deduced isoionic point of fromabout pH 10.1 to about pH 10.7.

[0012] The present invention also relates to peptides having an aminoacid sequence corresponding to an antigenic portion of the subjectchemokine, to antibodies which recognize this chemokine, and to isolatednucleic acid molecules which encode this chemokine.

[0013] The present invention also relates to an isolated nucleic acidmolecule which, under stringent conditions, hybridizes to a nucleic acidmolecule encoding a chemokine of the present invention or to acomplement thereof.

[0014] In another aspect thereof, the present invention relates to anisolated nucleic acid molecule which encodes for a chemokine of thepresent invention.

[0015] The present invention also relates to a method for detectingbreast disease in a patient. A sample of tissue or body fluid from thepatient is contacted with a nucleic acid primer which, under stringentconditions, hybridizes to a nucleic acid molecule encoding a chemokineof the present invention or to a complement thereof. The sample oftissue or body fluid from the patient in contact with the nucleic acidprimer is treated under conditions effective to amplify breast tissuespecific nucleic acid molecules. The method further includes detectingthe breast tissue specific nucleic acid molecules.

[0016] The present invention also relates to another method of detectingbreast disease in a patient. In this method, a sample of tissue or bodyfluid from the patient is contacted with a nucleic acid probe underconditions effective to permit formation of a hybridization complexbetween the probe and breast tissue specific nucleic acid molecules. Thenucleic acid probe is one which, under stringent conditions, hybridizesto a nucleic acid molecule encoding a chemokine of the present inventionor to a complement thereof. The method further includes detecting thehybridization complex.

[0017] The present invention also relates to yet another method ofdetecting breast disease in a patient. The method includes providing anantibody or binding portion thereof which recognizes a chemokine of thepresent invention. The antibody or binding portion thereof is contactedwith a liquid or tissue sample from the patient under conditionseffective to permit binding of the antibody or binding portion thereofto the chemokine in the liquid or tissue sample. The method furtherincludes detecting presence of antibody or binding portion thereof boundto the chemokine in the liquid or tissue sample.

[0018] The present invention, in another aspect thereof, relates to amethod of treating breast disease in a patient. The method includesadministering to the patient an effective amount of an antibody orbinding portion thereof which recognizes a chemokine of the presentinvention.

[0019] The present invention also relates to another method of treatingbreast disease in a patient. The method includes administering to thepatient an effective amount of a peptide which binds to a cellularreceptor for a chemokine of the present invention.

[0020] The present invention also relates to a method of vaccinating apatient against breast disease. The method includes administering to thepatient an effective amount of an antigenic portion of a chemokine ofthe present invention.

[0021] The chemokines, peptides, antibodies, and nucleic acid moleculesof the present invention are useful in the early detection of variouspathological states of the mammary gland, such as inflammations,infections, benign hyperplasias, and malignancies. In particular, theycan be used in the early detection of breast cancer as well as formonitoring the presence or absence of metastatic breast cancer cells ina patient's tissues and fluids, such as blood, lymph nodes, bone marrow,and other sites of disease dissemination. They can also be used to stagepatients with breast cancer and to assess the effects of conventionalbreast cancer therapies. Furthermore, the chemokines, peptides, andantibodies of the present invention can be used to treat or preventbreast disease.

BRIEF DESCRIPTION OF THE DRAWING

[0022]FIG. 1 is a series of aligned amino acid sequences of variousmembers of the CC chemokine family and the amino acid sequence of achemokine of the present invention. Members of the CC chemokine familythat are shown are HTECK (SEQ ID NO:20), LARC (SEQ ID NO:21), TARC (SEQID NO:22), I309 (SEQ ID NO:23), MCP-2 (SEQ ID NO:24), MCP-4 (SEQ IDNO:25), Eotaxin (SEQ ID NO:26), MCP-3 (SEQ ID NO:27), MCP-1 (SEQ IDNO:28), RANTES (SEQ ID NO:29), HCC-1 (SEQ ID NO:30), MIP-lB (SEQ IDNO:31), LB78B (SEQ ID NO:32), LD78A (SEQ ID NO:33), and PARC (SEQ IDNO:34).

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention relates to an isolated chemokine that ispreferentially expressed in breast tissue or that is detectable inbreast milk. Chemokine, as used herein, is meant to include proteinswhich are proinflammatory cytokines that are chemoattractants andactivators of specific types of leukocytes. Further details with respectto chemokine activity can be found, for example, in U.S. Pat. No.5,688,927 to Godiska et al. and Baggiolini et al., Advances inImmunology, 55:97-179 (1994), which are hereby incorporated by referenceThe chemokine may include a leader sequence, typically about 22 aminoacids in length, or, alternatively, the leader sequence can be cleavedfrom the chemokine. The isolated chemokine preferably includes fromabout 100 to 132 amino acids, more preferably, from about 105 to about127 amino acids, and, most preferably, about 105 or 127 amino acids. Thededuced molecular weight of the chemokine of the present invention ispreferably from about 10 to about 16 kDa, more preferably, from about 12kDa to about 14 kDa, and preferably has a deduced isoionic point of fromabout pH 10.1 to about pH 10.7, more preferably about 10.4.

[0024] As indicated above, the chemokine of the present invention ispreferentially expressed in breast tissue. That is, more chemokine ofthe present invention is expressed in breast tissue than in any othertissue in the body. More preferably, the chemokine of the presentinvention is expressed substantially exclusively or exclusively inbreast tissue. That is, substantially all of the chemokine of thepresent invention is expressed in breast tissue. In addition oralternatively to being preferentially expressed in breast tissue, thechemokine of the present invention can be detected in breast milk, suchas by using conventional protein detection methods.

[0025] One particularly preferred chemokine of the present invention hasan amino acid sequence corresponding to SEQ ID NO: 1, as follows:MQQRGLAIVALAVCAALHASEAILPIASSCCTEVSHHISRRLLERVNMCRIQRADGDCDLAAVILHVKRXRICVSPHNHTVKQWMKVQAAXKNGKGNVCHRKKHGKRNSNRAHQGKHETYGHKTPY

[0026] As indicated above, chemokine, as used herein, can include aleader sequence, or, alternatively, all or part of the leader sequencemay be removed. In SEQ ID NO: 1, approximately the first 22 amino acidsrepresents the leader sequence. Thus, chemokines of the presentinvention can also have an amino acid sequence corresponding to, forexample, SEQ ID NO: 2, as follows:LPIASSCCTEVSHHISRRLLERVNMCRIQRADGDCDLAAVILHVKRXRICVSPHNHTVKQWMKVQAAXKNGKGNVCHRKKHHGKRNSNRAHQGKHETYGH KTPY

[0027] The chemokine of the present invention is isolated (i.e.,substantially free of the biological materials with which it isnaturally found). In many applications, it is desirable that thechemokine of the present invention be purified (i.e., substantially freeof all other biological materials). The chemokines of the presentinvention can be in monomer form, or they can be associated with otherchemokines, such as in the form of dimers.

[0028] The present invention also relates to peptides which include anamino acid sequence corresponding to an antigenic portion of a chemokineof the present invention. In general, the size of the peptide antigen isnot believed to be particularly crucial, so long as it is at least largeenough to carry the antigenic core sequence or sequences. Generally, thesmallest useful antigenic sequence is on the order or about six aminoacids in length. However, the size of the antigen may be larger wheredesired, so long as it contains a basic antigenic core sequence.

[0029] Accordingly, through the use of computerized peptide sequenceanalysis program (DNAStar Software, DNAStar, Inc., Madison, Wis.), theportions of the peptide can be identified that are believed toconstitute antigenic sequences which include particular epitopes of theprotein. More particularly, antigenic portions of a chemokine of thepresent invention can be identified by hydropathy analysis, such as thatdescribed in Kyte et al., “A Simple Method for Displaying theHydropathic Character of a Protein,” J. Mol. Biol., 157:105-132 (1982),which is hereby incorporated by reference.

[0030] Synthesis of peptides which include an antigenic epitope withintheir sequence, are readily achieved using conventional synthetictechniques such as the solid phase method (e.g., through the use ofcommercially available peptide synthesizer such as an Applied BiosystemsModel 430A Peptide Synthesizer). Peptides synthesized in this manner maythen be aliquoted in predetermined amounts and stored in conventionalmanners, such as in aqueous solutions or, even more preferably, in apowder or lyophilized state pending use.

[0031] Particularly preferred peptides of the present invention arethose which include amino acid sequences corresponding toTEVSHHISRRLLERVNMC (SEQ ID NO: 3), KNGKGNVCHRKKHHGK (SEQ ID NO: 4), andNSNRAHQGKHETYGHKTPY (SEQ ID NO: 5).

[0032] As described below, the chemokines or peptides of the presentinvention can be used to raise antibodies that recognize chemokines ofthe present invention. The chemokines and peptides of the presentinvention can also be administered alone or in combination with apharmaceutically-acceptable carrier to patients, as a vaccine, forpreventing breast disease.

[0033] The present invention also relates to antibodies and bindingportions thereof which recognize a chemokine according to the presentinvention. Preferably, the antibody or binding portion thereof alsorecognizes particular antigenic portions of the subject chemokine, suchas peptides having amino acid sequences corresponding to SEQ ID NO: 3,SEQ ID NO: 4, and SEQ ID NO: 5.

[0034] The antibodies and binding portions thereof can be used to detectbreast disease in a patient. As used herein, breast disease is meant toinclude various pathological states of the mammary gland, such asinflammations, infections, mastitis, benign cystitis, benignhyperplasias, and cancer and other malignancies. Detection of breastdisease involves providing an antibody or binding portion thereof whichrecognizes a chemokine of the present invention. The antibody or bindingportion thereof is contacted with a tissue or fluid sample from thepatient under conditions effective to permit binding of the antibody orbinding portion thereof to chemokine that is present in the tissue orfluid sample to form a complex. The presence of a chemokine of thepresent invention in the tissue or fluid sample is detected by detectingthe complex.

[0035] Such contacting can be carried out in vivo in a living patient.In this embodiment of the present invention, the antibody or bindingportion thereof is administered (e.g., orally or parenterally) to thepatient under conditions effective to permit binding of the antibody orbinding portion thereof to the chemokine of the present invention in thein vivo tissue or fluid sample. Using this method, patients can bescreened for breast diseases associated with the presence of chemokinesof the present invention. Alternatively, the method can be used toidentify the recurrence of such diseases, particularly when the diseaseis localized in a particular biological material of the patient. Forexample, recurrence of breast disease in a patient's breast tissue canbe detected by administering a short range radiolabeled antibody to thepatient and then imaging the breast using conventional radiation imagingtechniques to detect the presence of the radiolabel and, therefore, aconcentration of a chemokine of the present invention, within thebreast. Similarly, by imaging other portions of the patient's body(e.g., lymph nodes), the method can be used to determine whether breastdisease (e.g., breast cancer) has spread to other tissues of the body.

[0036] Alternatively, the contacting step can be carried out in vitro.For example, the tissue or fluid sample can be a tissue specimen (e.g.,cells or tissue sections, preferably preserved by freezing or embeddingin paraffin, from the breast, lymph nodes, bone marrow, or other sitesof disease dissemination). Alternatively, the tissue or fluid sample canbe a fluid specimen (e.g., urine, serum, lymph fluid, and anticoagulatedwhole blood cells) removed from the patient.

[0037] The antibodies and binding portions thereof of the presentinvention can also be used to treat breast disease, for example, byablating or killing diseased breast tissue cells. The process involvesproviding an antibody or binding portions thereof which recognizes achemokine of the present invention. The antibody or binding portionsthereof can be used alone or can be bound to a substance effective tokill cells that are in proximity to an elevated level of a chemokine ofthe present invention or that bound to the chemokine. In this method,these antibodies or binding portions thereof are contacted with thecells under conditions effective to permit killing or ablating of thecells. In its preferred form, such contacting is carried out in a livingpatient by administering (e.g., orally or parenterally) the antibody orbinding portion thereof to the patient under conditions effective topermit localization of the antibody or binding portion thereof totissues having elevated concentrations of the subject chemokine andkilling or ablating of cells within such tissues.

[0038] Antibodies and binding portions thereof suitable for eitherkilling, ablating, or detecting diseased breast tissue cells includeantibodies, such as monoclonal or polyclonal antibodies. In addition,antibody fragments, half-antibodies, hybrid derivatives, and othermolecular constructs may be utilized. These antibodies and bindingportions recognize and bind to chemokines of the present invention,which are associated with breast disease.

[0039] Monoclonal antibody production may be effected by techniqueswhich are well-known in the art. Basically, the process involves firstobtaining immune cells (lymphocytes) from the spleen of a mammal (e.g.,mouse) which has been previously immunized with the antigen of interesteither in vivo or in vitro. The antibody-secreting lymphocytes are thenfused with (mouse) myeloma cells or transformed cells, which are capableof replicating indefinitely in cell culture, thereby producing animmortal, immunoglobulin-secreting cell line. The resulting fused cells,or hybridomas, are cultured, and the resulting colonies screened for theproduction of the desired monoclonal antibodies. Colonies producing suchantibodies are cloned and grown either in vivo or in vitro to producelarge quantities of antibody. A description of the theoretical basis andpractical methodology of fusing such cells is set forth in Kohler andMilstein, Nature 256:495 (1975), which is hereby incorporated byreference.

[0040] Mammalian lymphocytes are immunized by in vivo immunization ofthe animal (e.g., a mouse) with the protein or polypeptide of thepresent invention. Such immunizations are repeated as necessary atintervals of up to several weeks to obtain a sufficient titer ofantibodies. Following the last antigen boost, the animals are sacrificedand spleen cells removed.

[0041] Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is effected bystandard and well-known techniques, for example, by using polyethyleneglycol (“PEG”) or other fusing agents (see Milstein and Kohler, Eur. J.Immunol. 6:511 (1976), which is hereby incorporated by reference).

[0042] This immortal cell line, which is preferably murine, but may alsobe derived from cells of other mammalian species, including but notlimited to rats and humans, is selected to be deficient in enzymesnecessary for the utilization of certain nutrients, to be capable ofrapid growth, and to have good fusion capability. Many such cell linesare known to those skilled in the art, and others are regularlydescribed.

[0043] Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the protein orpolypeptide of the present invention subcutaneously to New Zealand whiterabbits which have first been bled to obtain pre-immune serum. Theantigens can be injected at a total volume of 100 μl per site at sixdifferent sites. Each injected material will contain adjuvants with orwithout pulverized acrylamide gel containing the protein or polypeptideafter SDS-polyacrylamide gel electrophoresis. The rabbits are then bledtwo weeks after the first injection and periodically boosted with thesame antigen three times every six weeks. A sample of serum is thencollected 10 days after each boost. Polyclonal antibodies are thenrecovered from the serum by affinity chromatography using thecorresponding antigen to capture the antibody. This and other proceduresfor raising polyclonal antibodies are disclosed in E. Harlow, et. al.,editors, Antibodies: A Laboratory Manual (1988), which is herebyincorporated by reference.

[0044] In addition to utilizing whole antibodies, the processes of thepresent invention encompass use of binding portions of such antibodies.Such binding portions include Fab fragments, F(ab′)₂ fragments, and Fvfragments. These antibody fragments can be made by conventionalprocedures, such as proteolytic fragmentation procedures, as describedin Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118,New York: Academic Press (1983), which is hereby incorporated byreference.

[0045] It is particularly preferred to use antibodies which recognize achemokine having an amino acid sequence corresponding to SEQ ID NO: 1 ora peptide having an amino acid sequence corresponding to SEQ ID NO: 3,SEQ ID NO: 4, or SEQ ID NO: 5. These antibodies can be used alone or asa component in a mixture with other antibodies or other biologicalagents to treat or image tissues containing a mammary associatedchemokine of the present invention.

[0046] Regardless of whether the antibodies or binding portions thereofare used for treatment or in vivo detection, they can be administeredorally, parenterally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, intraocularly, intraarterially,intralesionally, or by application to mucous membranes, such as, that ofthe nose, throat, and bronchial tubes. They may be administered alone orwith pharmaceutically or physiologically acceptable carriers,excipients, or stabilizers, and can be in solid or liquid form such as,tablets, capsules, powders, solutions, suspensions, or emulsions.

[0047] The solid unit dosage forms can be of the conventional type. Thesolid form can be a capsule, such as an ordinary gelatin type containingthe antibodies or binding portions thereof of the present invention anda carrier, for example, lubricants and inert fillers such as, lactose,sucrose, or cornstarch. In another embodiment, these compounds aretableted with conventional tablet bases such as lactose, sucrose, orcornstarch in combination with binders like acacia, cornstarch, orgelatin, disintegrating agents, such as cornstarch, potato starch, oralginic acid, and a lubricant, like stearic acid or magnesium stearate.

[0048] The antibody or binding portion thereof of the present inventionmay also be administered in injectable dosages by solution or suspensionof these materials in a physiologically acceptable diluent with apharmaceutical carrier. Such carriers include sterile liquids, such aswater and oils, with or without the addition of a surfactant and otherpharmaceutically and physiologically acceptable carrier, includingadjuvants, excipients or stabilizers. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solution, and glycols, such as propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions.

[0049] For use as aerosols, the antibody or binding portion thereof ofthe present invention in solution or suspension may be packaged in apressurized aerosol container together with suitable propellants, forexample, hydrocarbon propellants like propane, butane, or isobutane withconventional adjuvants. The materials of the present invention also maybe administered in a non-pressurized form such as in a nebulizer oratomizer.

[0050] As indicated above, the antibody or binding portion thereof maybe used to detect, in vivo, breast disease in a patient. This ispreferably achieved by labeling the antibody or binding portion thereof,administering the labeled antibody or binding portion thereof to thepatient, and then imaging the patient.

[0051] Examples of labels useful for diagnostic imaging in accordancewith the present invention are radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I,⁹⁹mTc, ³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such asfluorescein and rhodamine, nuclear magnetic resonance active labels,positron emitting isotopes detectable by a positron emission tomography(“PET”) scanner, chemiluminescers such as luciferin, and enzymaticmarkers such as peroxidase or phosphatase. Short-range radiationemitters, such as isotopes detectable by short-range detector probes canalso be employed. The antibody or binding portion thereof can be labeledwith such reagents using techniques known in the art. For example, seeWensel and Meares, Radioimmunoimaging and Radioimmunotherapy, New York:Elsevier (1983), which is hereby incorporated by reference, fortechniques relating to the radiolabeling of antibodies. See also,Colcher et al., “Use of Monoclonal Antibodies as Radiopharmaceuticalsfor the Localization of Human Carcinoma Xenografts in Athymic Mice”,Meth. Enzymol. 121:802-816 (1986), which is hereby incorporated byreference.

[0052] Detecting the presence of a complex between an antibody orbinding portion thereof and a chemokine of the present invention can becarried out by any conventional method for detecting antigen-antibodyreactions, examples of which can be found, e.g., in Klein, Immunology,New York: John Wiley & Sons, pp. 394-407 (1982), which is herebyincorporated by reference. For in vitro detection of breast disease, theformation of a complex between the antibody and chemokine present in thetissue of fluid sample can be detected by enzyme linked assays, such asELISA assays. Briefly, the antibody/chemokine complex is contacted witha second antibody which recognizes a portion of the antibody that iscomplexed with the chemokine. Generally, the second antibody is labeledso that its presence (and, thus, the presence of an anntibody/chemokinecomplex) can be detected. Alternatively, the antibody or binding portionthereof can be bound to a label effective to permit detection of thechemokine upon binding of the antibody or binding portion thereof to thechemokine. Suitable labels include, fluorophores, chromophores,radiolabels, and the like.

[0053] For example, a radiolabeled antibody or binding portion thereofof this invention can be used for in vitro diagnostic tests. Thespecific activity of a tagged antibody or binding portion thereofdepends upon the half-life and isotopic purity of the radioactive labeland how the label is incorporated into the antibody or its bindingportion. Table 1 lists several commonly-used isotopes, their specificactivities and half-lives. In immunoassay tests, the higher the specificactivity, in general, the better the TABLE 1 Specific Activity of PureIsotope Isotope (Curies/mole) Half-Life ¹⁴C 6.25 × 10¹ 5720 years ³H2.01 × 10⁴  12.5 years ³⁵S 1.50 × 10⁶  87 days ¹²⁵I 2.18 × 10⁶  60 days³²P 3.16 × 10⁶  14.3 days ¹³¹I 1.62 × 10⁷   8.1 days

[0054] Procedures for labeling antibodies and binding portions thereofwith the radioactive isotopes listed in Table 1 are generally known inthe art. Tritium labeling procedures are described in U.S. Pat. No.4,302,438 to Zech, which is hereby incorporated by reference.Iodinating, tritium labeling, and 35S labeling procedures especiallyadapted for murine monoclonal antibodies are described in Goding,Monoclonal Antibodies: Principles and Practice, pp. 124-126, New York:Academic Press (1983) and the references cited therein, which are herebyincorporated by reference. Other procedures for iodinating antibodies orbinding portions thereof are described in Hunter et al., Nature 144:945(1962), David et al., Biochemistry 13:1014-1021 (1974), U.S. Pat. No.3,867,517 to Ling, and U.S. Pat. No. 4,376,110 to David et al., whichare hereby incorporated by reference. Radiolabeling elements which areuseful in imaging include ¹²³I, ¹³¹I, ¹¹¹In, and ^(99m)Tc, for example.Procedures for iodinating antibodies or binding portions thereof aredescribed in Greenwood et al., Biochem. J. 89:114-123 (1963);Marchalonis, Biochem. J. 113:299-305 (1969); and Morrison et al.,Immunochemistry 289-297 (1971), which are hereby incorporated byreference. Procedures for ^(99m)Tc-labeling are described by Rhodes etal. in Burchiel et al., eds., Tumor Imaging: The RadioimmunochemicalDetection of Cancer, New York: Masson 111-123 (1982) and the referencescited therein, which are hereby incorporated by reference. Proceduressuitable for ¹¹¹In-labeling antibodies or binding portions thereof aredescribed by Hnatowich et al., J. Immul. Methods 65:147-157 (1983),Hnatowich et al., J. Applied Radiation 35:554-557 (1984), and Buckley etal., F.E.B.S. 166:202-204 (1984), which are hereby incorporated byreference.

[0055] The antibodies or binding portions thereof of the presentinvention can be used and sold together with equipment to detect theparticular label as a kit for in vitro detection of breast disease.

[0056] In the case of a radiolabeled antibody or binding portionthereof, the antibody or binding portion thereof is administered to thepatient, is localized to the region of the patient where diseased breastcells produce increased levels of the subject chemokines, and isdetected or “imaged” in vivo using known techniques such as radionuclearscanning using e.g., a gamma camera or emission tomography. See e.g.,Bradwell et al., “Developments in Antibody Imaging” in Baldwin et al.,eds., Monoclonal Antibodies for Cancer Detection and Therapy, pp. 65-85,New York: Academic Press (1985), which is hereby incorporated byreference. Alternatively, a positron emission transaxial tomographyscanner, such as the one designated Pet VI located at BrookhavenNational Laboratory, can be used where the radiolabel emits positrons(e.g., ¹¹C, ¹⁸F, ¹⁵O, and ¹³N) Fluorophore and chromophore labeledantibodies and binding portions thereof can be prepared from standardmoieties known in the art. Since antibodies and other proteins absorblight having wavelengths up to about 310 nm, the fluorescent moietiesshould be selected to have substantial absorption at wavelengths above310 nm and preferably above 400 nm. A variety of suitable fluorescersand chromophores are described in Stryer, Science, 162:526 (1968) andBrand et al., Annual Review of Biochemistry, 41:843-868 (1972), whichare hereby incorporated by reference. The antibodies and bindingportions thereof can be labeled with fluorescent chromophore groups byconventional procedures such as those disclosed in U.S. Pat. No.3,940,475 to Gross, U.S. Pat. No. 4,289,747 to Chu, and U.S. Pat. No.4,376,110 to David et al., which are hereby incorporated by reference.

[0057] One group of fluorescers having a number of the desirableproperties described above are the xanthene dyes, which include thefluoresceins derived from 3,6-dihydroxy-9-hexylxanthhydrol and resaminesand rhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanimerhodamine B. The rhodamine and fluorescein derivatives of9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluoresceincompounds having reactive coupling groups such as amino andisothiocyanate groups such as fluorescein isothiocyanate andfluorescamine are readily available. Another group of fluorescentcompounds are the naphthylamines, having an amino group in the α or βposition.

[0058] Antibodies and binding portions thereof can be labeled withfluorchromes or chromophores by the procedures described in Goding,Monoclonal Antibodies: Principles and Practice, pp. 208-249, New York:Academic Press (1983), which is hereby incorporated by reference. Theantibodies and binding portions thereof can be labeled with anindicating group containing the NMR-active ¹⁹F atom, or a plurality ofsuch atoms inasmuch as (i) substantially all of naturally abundantfluorine atoms are the ¹⁹F isotope and, thus, substantially allfluorine-containing compounds are NMR-active; (ii) many chemicallyactive polyfluorinated compounds such as trifluoracetic anhydride arecommercially available at relatively low cost, and (iii) manyfluorinated compounds have been found medically acceptable for use inhumans such as the perfluorinated polyethers utilized to carry oxygen ashemoglobin replacements. After permitting such time for incubation, awhole body NMR determination is carried out using an apparatus such asone of those described in Pykett, Scientific American, 246:78-88 (1982),which is hereby incorporated by reference, to locate and image regionsof elevated chemokine concentration.

[0059] The antibodies and binding portions thereof can also be utilizedto treat breast disease in vivo. This involves administering to apatient in need of such treatment the antibodies or binding portionsthereof by themselves or with a cytotoxic drug to which the antibodiesand binding portions thereof are bound. Since the antibodies and bindingportions thereof recognize the subject chemokines, diseased breastcells, which are in proximity to elevated levels of the subjectchemokines which they produce, are destroyed. Caution must be exercised,however, as such administration may destroy normal cells which are inproximity to the chemokines produced by the diseased breast cells.

[0060] The antibodies and binding portions thereof of the presentinvention may be used to deliver a variety of cytotoxic drugs includingtherapeutic drugs, a compound emitting radiation, molecules of plants,fungal, or bacterial origin, biological proteins, and mixtures thereof.

[0061] Enzymatically active toxins and fragments thereof are exemplifiedby diphtheria toxin A fragment, nonbinding active fragments ofdiphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin Achain, abrin A chain, modeccin A chain, α-sacrin, certain Aleuritesfordii proteins, certain Dianthin proteins, Phytolacca americanaproteins (PAP, PAPII and PAP-S), Morodica charantia inhibitor, curcin,crotin, Saponaria officinalis inhibitor, gelonin, mitogillin,restrictocin, phenomycin, and enomycin, for example. Procedures forpreparing enzymatically active polypeptides of the immunotoxins aredescribed in WO84/03508 and WO85/03508, which are hereby incorporated byreference. Certain cytotoxic moieties are derived from adriamycin,chlorambucil, daunomycin, methotrexate, neocarzinostatin, and platinum,for example.

[0062] Procedures for conjugating the antibodies and binding portionsthereof with the cytotoxic agents have been previously described.Procedures for conjugating chlorambucil with antibodies are described inFlechner, European Journal of Cancer 9:741-745 (1973); Ghose et al.,British Medical Journal 3:495-499 (1972); and Szekerke et al., Neoplasma19:211-215 (1972), which are hereby incorporated by reference.Procedures for conjugating daunomycin and adriamycin to antibodies aredescribed in Hurwitz et al., Cancer Research 35:1175-1181 (1975) andArnon et al. Cancer Surveys 1:429-449 (1982), which are herebyincorporated by reference. Procedures for preparing antibody-ricinconjugates are described in U.S. Pat. No. 4,414,148 to Jansen et al. andin Osawa et al. Cancer Surveys 1:373-388 (1982) and the references citedtherein, which are hereby incorporated by reference. Coupling proceduresare also described in EP 86309516.2, which is hereby incorporated byreference.

[0063] The use of the subject antibodies and binding portions thereofcan also be used in a drug/prodrug treatment regimen. In this method,for example, a first antibody or binding portion thereof according tothe present invention is conjugated with a prodrug which is activatedonly when in close proximity with a prodrug activator. The prodrugactivator is conjugated with a second antibody or binding portionthereof, preferably one which binds to diseased breast cells or to otherbiological materials associated with diseased breast cells (e.g.,another protein produced by diseased breast cells). Drug-prodrug pairssuitable for use in the practice of the present invention are describedin Blakely et al., “ZD2767, an Improved System for Antibody-directedEnzyme Prodrug Therapy That Results in Tumor Regressions in ColorectalTumor Xenografts,” Cancer Research 56:3287-3292 (1996), which is herebyincorporated by reference.

[0064] Alternatively, the antibody or binding portion thereof can becoupled to high energy radiation emitters, for example, a radioisotope,such as ¹³¹I, a γ-emitter, which, when localized at the diseased breasttissue site, results in a killing of several cell diameters. See, e.g.,Order, “Analysis, Results, and Future Prospective of the Therapeutic Useof Radiolabeled Antibody in Cancer Therapy” in Baldwin et al., eds.,Monoclonal Antibodies for Cancer Detection and Therapy, pp 303-316, NewYork: Academic Press (1985), which is hereby incorporated by reference.Other suitable radioisotopes include α-emitters, such as ²¹²Bi, ²¹³Bi,and ²¹¹At, and β-emitters, such as ¹⁸⁶Re and ⁹⁰Y.

[0065] Where the antibodies or binding portions thereof are used aloneto treat breast disease, such treatment can be effected by initiatingendogenous host immune functions, such as complement-mediated orantibody-dependent cellular cytotoxicity.

[0066] The antibodies or binding portions thereof of the presentinvention can be used in conjunction with other therapeutic treatmentmodalities. Such other treatments include surgery, radiation,cryosurgery, thermotherapy, hormone treatment, chemotherapy, vaccines,and other immunotherapies.

[0067] Also encompassed by the present invention is a method of treatingbreast disease which involves using the antibodies and binding portionsthereof without cytotoxic agents for prophylaxis. For example, theantibodies and binding portions thereof can be used to prevent or delaydevelopment or progression of breast disease by binding to thechemokines of the present invention and, thus, inhibiting theirbiological activity.

[0068] Another aspect of the present invention relates to an isolatednucleic acid molecule which encodes a chemokine of the presentinvention. The encoded chemokine is preferably one that ispreferentially expressed in breast tissue or one which can be detectedin breast milk. The encoded chemokine can include from about 100 toabout 132 amino acids, preferably from about 105 to about 127 aminoacids, more preferably, about 105 or 127 amino acids; can have a deducedmolecular weight of from about 10 to about 16 kDa, preferably from about12 kDa to about 14 kDa; and can have a deduced isoionic point of fromabout pH 10.1 to about pH 10.7, preferably about 10.4. The term“isolated nucleic acid molecules” is intended to refer to nucleic acidmolecules that are substantially free of the biological materials withwhich they are naturally found. The term “nucleic acid” is meant torefer to polydeoxyribonucleotides (“DNA”), which contain2-deoxy-D-ribose, to polyribonucleotides (“RNA”), which containD-ribose, and to any other type of polynucleotide which is anN-glycoside of a purine or pyrimidine base or a modified purine orpyrimidine base. The term “nucleic acid” refers only to the primarystructure of the molecule, and, thus, it is meant to include double- andsingle-stranded DNA as well as double- and single-stranded RNA. There isno intended distinction in length between the terms “nucleic acid” and“oligonucleotide”, and these terms are used interchangeably herein.

[0069] The nucleic acid molecule can be a DNA or RNA molecule whichencodes a chemokine having an amino acid sequence corresponding to SEQID NO: 1. One such nucleic acid molecule has a nucleotide sequencecorresponding to SEQ ID NO: 6 as follows: AACATCCTCA CTTGTGTTGCTGTCAGTGCC TGTANGGCAG GCAGGAATGC AGCAGAGAGG ACTCGCCATC GTGGCCTTGGCTGTCTGTGC GGCCCTACAT GCCTCAGAAG CCATACTTCC CATTGCCTCC AGCTGTTGCACGGAGGTTTC ACATCATATT TCCAGAAGGC TCCTGGAAAG AGTGAATATG TGTCGCATCCAGAGAGCTGA TGGGGATTGT GACTTGGCTG CTGTCATCCT TCATGTCAAG CGCNGAAGAATCTGTGTCAG CCCGCACAAC CATACTGTTA AGCAGTGGAT GAAAGTGCAA GCTGCCAANAAAAATGGTAA AGGAAATGTT TGCCACAGGA AGAAACACCA TGGCAAGAGG AACAGTAACAGGGCACATCA GGGGAAACAC GAAACATACG GCCATAAAAC TCCTTATTAG AGAATCTACAGATAAATCTA CAGAGACAAT CCCCCAAGTG GACTTGGCCA TGATTGGTTG TAAGTTTATCATCTGAATTC TCCTTATTGT AGACAACAGA ACAAAACAAA ATATTGGTTT TTAAAAAATGAACAATTGTG CCGTATGCAA ATGTACCCAA TAATATACTC CACTGGAAAA TGAAATGAAAAAANNATACT GGCTGGGTAT GGTGGGTCCC CCCTTTTATC CCANNNNCTT CGGGAGGCAGAGGCAGGAGG ATCACTTGAG ACCAGGANTT NGAGACNAGC TNGGGGCAAA ANAGCAANGACNTCATTTNT ACAAACNAAA AAAAANNTTG GCCCGGCNTG GTAGNACTTG CNTATAATCCCAGCNACATG GGAGGTNGAG GTGGGAGGAT CACTTGAGTC TGGGNGAGTT NGAGGTNGCAGTGAGCAGCN TGGGTGACAG AATGNAGACC NTGTCTCTAA AAATAATAAT AATAATGATAGTGTATATCT TCATATAATA TTTTAAGNAG GAGCATATAG ATATAACTTN CTCCCAACTTTTTAATTATA GTTTTCCAAA CTTACAGAGA AGTTAAAAGA ATGGTACAAT GAACATCTATATATCTTTCA CCACAATATT AATCATTGTT AATATTGTGC CACATTTGCT TTCTCTCTCCTCTCTTGGTA GGGGTTNCAA TATAAAATAT TATAACTTTT AAAATATATC TTGTTTTGCTAACCATTGGA AAATAAGTTG CAAAAATCAT GACACTTCAC CCCTAGTTTC TTTTNGGTGTTATAACTTGA CATACCCTAA AATAAAGACA TTTTTCTACA TAATCACCTT ATCAGTTTTATACCTAAAAA ATTAATAATT TCATCTAATA TATTCCATAT TCAAATTTTC CCAACTATTTAGAGAGCATT TTATGTAGTT TTTTTTTCAC TCCAGTAATC AATCAAGGTN GACATACATATTGCAAATAA TTGTTATTTT TCTTTAATAT CTTTCAATCT AAGAAAGTTC CTCTGTCTTTTTTTTTTAAT TTTTAAAATT ATTTTGTTGA GGGAGGGTCT TGCTGTGTCT TCCAGGCTGGAGTGCAGTGG CACAATTTTG ATTTTGGCTC ACTGAAGCCT CAACTTTAGG GCTCAAGCAATCCTCCCACC TCAGCCTNCC CGAGTATCTG GGATCAAGGT GCATACCCAC CACACCTGGCTAATTTTGTT TATTTTTTGT AGAGACAGGG TCTCACTATG TTGCCCAGGT TGATCTCAAACTCCTGGGCT CAAGCGATCC TCCCACCTTA GCCTCCCAAA GTACTGGGAT TATAGGTGTGAGCCACAGTG CCTGGCCTAA TTATTTTCTT GTGATCAAAT TCAGGTTTAA TGTTTTTGGTTAAGAATTTC CTACGTGAAT TCGTGTACTT ATTTTGTCAT TTAGAGTTCA TAAATATTAGGGTTTATTTT CTAAATAGAA TAGTTTAAAC TAAATATAAC TTCAAAACGT CTAGTTTGAGTAGCTACCGT TGTTTGGATT GAAATTTTCT GATACTGAAA AGAACAAAAA GCCTGCCTTTCTGCCCANAA CSNNTTGCYT CCCCCAGTNA GTTCTTGGNG CAGNACTAGT TAGGGNCCCAGAGTTNGGCC TTNNGKGTGG TGATTTTANG YTCTGCCTAA ACAAGGNGCN WACATYTTTTAGCTCCTATT CCACCYTTCT NAMAMGTTTT TGTTGTKGTT TGNTTGTTTT TTTKGAGACAGRRTNTNAYT CTGTTTGCCC ARGCTGGART TGCAGTGGCA CAATYTNGGY TNCATTGCAACYTCNGCYTC CSSGCCGTTC AAKTGATYYT CTTGCYTCAG CYTCCCCAAG TAANTGATATTACAGGNGCC CAGCCACCAM ACCCCGNTGA WTTTTGTATT TTTARTARAR AMRGGGTTTTCCCGCNTTGG CNGGGCTGGT CTCNAANTCC TTGAMCTCNA KTGAACCACC CGCCTGTGCCYCCCAAANTG CTGGAATTAC CANCGTTGAN CCACCATGCC GGGCYCACAC GTTTGARTTTGANACCATTG TNCCATTCCT CTTTTGGCCT YTTTTTTNTC CATAGNNGCT TCAAGATAGATANGTAAGRG CCCAGTAGTN GTTCWTARGA AGCNMATAGR RANCRGGARC CANTTTNATCAGGTGGGCAG GTGTCCNNGG CYTCCCTGCT GGYTNNTCCC AAGCGGTGGT GTTGCCARGANKTNTTGGAR GTGATAATGG GANANACCAG NAGGCMCTGA GTYNCNNTAG GTTNAAATGCCACCAAAACT GGCCTTTGGC CTAATATCCY YCNTTGAMTA NTTARCATTT AWTTTATTWATTTNCCTGAC ATTTNTGCMA NCCTTTGTWT TTNTATTTCC NCTNTATARA WGARGAAATTTGAGGNTYTT ARAGGTAAAA TGANTTGCNC NRGTNNACMC AGGAAGTGGC NRARANAANCTTTTTANATN MGAAAAAATT AATAAAATAT AATATGAGAG TAACTTAAAA TATTAATAAACCACAATTTT AAATTAATTA ACCGTGATAA CCAACATTAA TAAAAGTTAA GATACCAAAACACTGGTGTN TAATTTTTTN AACTAACAAN TTGAATTATT TTCCATTTTA AATTAATTAACCGTGATAAC CAACATTAAT AAAAGTTAAG ATACCGN

[0070] Another such nucleic acid molecule has a nucleotide sequencecorresponding to SEQ ID NO: 7 as follows: ATGCAGCAGA GAGGACTCGCCATCGTGGCC TTGGCTGTCT GTGCGGCCCT ACATGCCTCA GAAGCCATAC TTCCCATTGCCTCCAGCTGT TGCACGGAGG TTTCACATCA TATTTCCAGA AGGCTCCTGG AAAGAGTGAATATGTGTCGC ATCCAGAGAG CTGATGGGGA TTGTGACTTG GCTGCTGTCA TCCTTCATGTCAAGCGCNGA AGAATCTGTG TCAGCCCGCA CAACCATACT GTTAAGCAGT GGATGAAAGTGCAAGCTGCC AANAAAAATG GTAAAGGAAA TGTTTGCCAC AGGAAGAAAC ACCATGGCAAGAGGAACAGT AACAGGGCAC ATCAGGGGAA ACACGAAACA TACGGCCATA AAACTCCTTA T

[0071] This nucleic acid represents an open reading frame of the nucleicacid molecule having a nucleotide sequence corresponding to SEQ ID NO:6.

[0072] The above isolated nucleic acid molecules of the presentinvention which encode for chemokines of the present invention can beused along with conventional recombinant methods to produce isolatedchemokines of the present invention

[0073] Briefly, this is carried out by incorporating any one of the DNAmolecules encoding chemokines of the present invention in cells usingconventional recombinant DNA technology. This involves inserting theselected DNA molecule into an expression system to which that DNAmolecule is heterologous (i.e., not normally present). The heterologousDNA molecule is inserted into the expression system or vector in properorientation and correct reading frame. The vector contains the necessaryelements for the transcription and translation of the insertedprotein-coding sequences.

[0074] U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is herebyincorporated by reference, describes the production of expressionsystems in the form of recombinant plasmids using restriction enzymecleavage and ligation with DNA ligase. These recombinant plasmids arethen introduced by means of transformation and replicated in unicellularcultures including procaryotic organisms and eucaryotic cells grown intissue culture.

[0075] Recombinant genes may also be introduced into viruses, such asvaccina virus. Recombinant viruses can be generated by transfection ofplasmids into cells infected with virus.

[0076] Suitable vectors include, but are not limited to, the followingviral vectors such as lambda vector system gt11, gt WES.tB, Charon 4,and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8,pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript IISK +/− or KS +/− (see “Stratagene Cloning Systems” Catalog (1993) fromStratagene, La Jolla, Calif, which is hereby incorporated by reference),pQE, pIH821, pGEX, pET series (see Studier et. al., “Use of T7 RNAPolymerase to Direct Expression of Cloned Genes” in Gene ExpressionTechnology, vol. 185 (1990), which is hereby incorporated by reference)and any derivatives thereof. Recombinant molecules can be introducedinto cells via transformation, particularly transduction, conjugation,mobilization, or electroporation. The DNA sequences are cloned into thevector using standard cloning procedures in the art, as described byManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor, N.Y.: Cold Springs Laboratory Press (1982), which is herebyincorporated by reference.

[0077] A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus). The expression elements of these vectors varyin their strength and specificities. Depending upon the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements can be used.

[0078] Different genetic signals and processing events control manylevels of gene expression (e.g., DNA transcription and messenger RNA(mRNA) translation).

[0079] Transcription of DNA is dependent upon the presence of a promoterwhich is a DNA sequence that directs the binding of RNA polymerase andthereby promotes mRNA synthesis. The DNA sequences of eucaryoticpromoters differ from those of procaryotic promoters. Furthermore,eucaryotic promoters and accompanying genetic signals may not berecognized in or may not function in a procaryotic system, and, further,procaryotic promoters are not recognized and do not function ineucaryotic cells.

[0080] Similarly, translation of mRNA in procaryotes depends upon thepresence of the proper procaryotic signals which differ from those ofeucaryotes. Efficient translation of mRNA in procaryotes requires aribosome binding site called the Shine-Dalgamo (“SD”) sequence on themRNA. This sequence is a short nucleotide sequence of mRNA that islocated before the start codon, usually AUG, which encodes theamino-terminal methionine of the protein. The SD sequences arecomplementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probablypromote binding of mRNA to ribosomes by duplexing with the rRNA to allowcorrect positioning of the ribosome. For a review on maximizing geneexpression, see Roberts et al., Methods in Enzymology 68:473 (1979),which is hereby incorporated by reference.

[0081] Promoters vary in their “strength” (i.e., their ability topromote transcription). For the purposes of expressing a cloned gene, itis desirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

[0082] Bacterial host cell strains and expression vectors may be chosenwhich inhibit the action of the promoter unless specifically induced. Incertain operons, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

[0083] Specific initiation signals are also required for efficient genetranscription and translation in procaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ tothe initiation codon (ATG) to provide a ribosome binding site. Thus, anySD-ATG combination that can be utilized by host cell ribosomes may beemployed. Additionally, any SD-ATG combination produced by recombinantDNA or other techniques involving incorporation of synthetic nucleotidesmay be used.

[0084] Once the desired isolated DNA molecule encoding a chemokineaccording to the present invention has been cloned into an expressionsystem, it is ready to be incorporated into a host cell. Suchincorporation can be carried out by the various forms of transformationnoted above, depending upon the vector/host cell system. Suitable hostcells include, but are not limited to, bacteria, virus, yeast, mammaliancells, and the like.

[0085] Recombinant DNA technology can also be used to produce fragmentsof the above chemokines, such as the above-referenced peptides. Forexample, subclones of the gene encoding a subject chemokine are producedby conventional molecular genetic manipulation by subcloning genefragments. The subclones then are expressed in vitro or in vivo inbacterial cells to yield a smaller peptide that can be tested for itsantigenic activity (i.e., capacity to be used as an antigen to raiseantibodies which recognize an antigenic portion of the chemokine).

[0086] As an alternative, protein fragments can be produced by digestionof a full-length subject chemokine with proteolytic enzymes likechymotrypsin, Staphylococcus proteinase A, or trypsin. Differentproteolytic enzymes are likely to cleave proteins at different sitesbased on the amino acid sequence of the protein. Some of the fragmentsthat result from proteolysis may have antigenic activity.

[0087] In still another approach, based on knowledge of the primarystructure of the subject chemokines, fragments of the encoding gene maybe synthesized by using the polymerase chain reaction (“PCR”) techniquetogether with specific sets of primers chosen to represent particularportions of the protein. These then would be cloned into an appropriatevector to facilitate expression of a peptide having, for example,antigenic activity.

[0088] Chemical synthesis can also be used to make suitable fragments.Such a synthesis is carried out using known amino acid sequences for thechemokines of the present invention. Alternatively, subjecting a fulllength subject chemokine to high temperatures and pressures will producefragments. These fragments can then be separated by conventionalprocedures (e.g., chromatography and SDS-PAGE).

[0089] The chemokines of the present invention and their fragments canoptionally be modified by, for example, the deletion or addition ofamino acids that have minimal influence on the properties, secondarystructure, and hydropathic nature of the chemokine or fragments. Forexample, a chemokine or peptide of the present invention can beconjugated to a signal (or leader) sequence at the N-terminal end of thechemokine which co-translationally or post-translationally directstransfer of the protein. The chemokine or peptide can also be conjugatedto a linker or other sequence for ease of protein synthesis,purification, or identification. The peptides of the present inventioncan also include, in addition to the antigenic portion of the chemokine,other amino acid sequences, such as T-cell antigenic stimuli and otheramino acid sequences which increase the peptide's immunogenicity.

[0090] As indicated above, the chemokines and peptides of the presentinvention are preferably produced in purified form (preferably at leastabout 80%, more preferably 90% pure) by conventional techniques. Thechemokines or peptides of the present invention are preferably producedin purified form by conventional techniques, of which the following isone example. To isolate the proteins, an E. coli host cell carrying arecombinant plasmid is propagated and homogenized, and the homogenate iscentrifuged to remove bacterial debris. The supernatant is thensubjected to sequential ammonium sulfate precipitation. The fractioncontaining the chemokines or peptides of the present invention issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the chemokines or peptides. Ifnecessary, the chemokine or peptide fraction may be further purified byion exchange chromatography and/or HPLC.

[0091] As indicated above, the chemokines and peptides of the presentinvention can be used to raise antibodies which are useful in thedetection and treatment of breast disease. Breast disease can also betreated using the peptides of the present invention by administering toa patient suffering from breast disease an effective amount of a peptidewhich binds to a cellular receptor for a chemokine of the presentinvention. Methods for identifying peptides which bind to cellularreceptors of proteins having known amino acid sequences are well knownto those skilled in the art and are described in, for example, Wells etal., “Selectivity and Antagonism of Chemokine Receptors,” J. LeukocyteBiol., 59:53-60 (1996) and Horuk, “Molecular Properties of the ChemokineReceptor Family,” Trends Pharmacol. Sci., 15:159-165 (1994), which arehereby incorporated by reference.

[0092] The present invention also relates to isolated nucleic acidmolecules which, under stringent conditions, hybridize to a nucleic acidmolecule encoding a chemokine of the present invention. Such isolatednucleic acid molecules include those which hybridize, under stringenthybridization conditions, to nucleic acid molecules (1) which encodechemokines that are preferentially expressed in breast tissue or thatare detected in breast milk; (2) which encode chemokines which includefrom about 100 to about 132 amino acids, which have a deduced molecularweight of from about 10 to about 16 kDa, and which have a deducedisoionic point of from about pH 10.1 to about pH 10.7; (3) which encodechemokines which include from about 105 to about 127 amino acids, whichhave a deduced molecular weight of from about 12 to about 14 kDa, andwhich have an isoionic point of about pH 10.4; (4) which encodechemokines having an amino acid sequence corresponding to SEQ ID NO: 1;(5) which have a nucleotide sequence corresponding to SEQ ID NO:6; and(6) which have a nucleotide sequence corresponding to SEQ ID NO:7.Preferably, the nucleic acid molecules which hybridize under stringentconditions to nucleic acid molecules encoding a chemokine of the presentinvention preferentially hybridize to nucleic acid molecules from breasttissue. That is, more of the chemokine of the present invention willhybridize, under stringent conditions, to nucleic acid molecules frombreast tissue that to nucleic acid molecules from other tissues in thebody.

[0093] The present invention also relates to isolated nucleic acidmolecules which, under stringent conditions, hybridize to the complementof a nucleic acid molecule encoding a chemokine of the presentinvention.

[0094] “Stringent conditions”, as used herein in relation tohybridization, mean approximately 35° C. to 70° C., preferably about 50°C., 55° C., 60° C., and/or 65° C., in a salt solution of approximately0.9 molar NaCl. These conditions are frequently represented by a washstringency of 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS at 70° C. to aDNA molecule encoding a chemokine of the present invention in a standardin situ hybridization assay. See Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory (1989). In general, such sequences will be at least 95%homologous, often at least 98% homologous, and even at least 99%homologous with the sequences of DNA molecules encoding chemokines ofthe present invention.

[0095] Illustrative nucleic acid molecules include those which have anucleotide sequence corresponding toACACGAATTCACGTAGGAAATTCTTAACCAAAAACATTAAACCTGAATTTGATCACAAGAAAATAATTAGGCCAGGCACTGTGGCTCACACCTATAATCCCAGT(SEQ ID NO:8), GAATTCACGTAGGAA ATTCTTAACC (SEQ ID NO:9),ACTGGGATTATAGGTGTGAGCC (SEQ ID NO:10), andGGAGAGAGCCGTATGTTTCGTGTTTCCCCTGATGTGCCCTGTTACTGTTCCTCTTGCCATGGTGTTTCTTCCTGTGGCAAACATTTCCTTTACCATTTTTNTTGGCAGCTTGCACTTTCATCCACTGCTTAACAGTATGGTTGTGCGGGCTGACACAGATTNTTCTGCGCTTGACATGAAGGATGACAGCAGCCAAGTCACAATCCCCATCAGCTCTCTGGATGCGACACATATTCACTCTTTCCAGGAGCCTTCTGGAAATATGATGTGAAACCTCCGTGCAACAGCTGGAGGCAATGGGAAGTATGGCT(SEQ ID NO: 11), as well as to those which have a nucleotide sequencecorresponding to a complement of and of SEQ ID NOs:. 8-11. Of course, asone skilled in the art will recognize, although these exemplary nucleicacid molecules have a defined number of nucleotides, one or morenucleotides may be added or deleted from a particular nucleic acidmolecule without great impact on its ability to hybridize with a nucleicacid molecule encoding a chemokine of the present invention.

[0096] The exact size of nucleic acid molecules which hybridize understringent conditions to nucleic acid molecules encoding a chemokine ofthe present invention depends on many factors and the ultimate use towhich the nucleic acid molecule is to be put. These nucleic acidmolecules can be prepared by any suitable method, such as by cloning andrestriction of appropriate sequences and by direct chemical synthesisusing, for example, the phosphotriester method (described in, e.g.,Narang et al., Meth. Enzymol. 68:90-99 (1979), which is herebyincorporated by reference); the phosphodiester method (described in,e.g., Brown et al., Meth. Enzymol. 68:109-151 (1979), which is herebyincorporated by reference); the diethylphosphoramidite method (describedin, e.g., Beaucage et al., Tetrahedron Lett. 22:1859-1862 (1981), whichis hereby incorporated by reference); and the solid support method(described in, e.g., U.S. Pat. No. 4,458,066 to Caruthers et al., whichis hereby incorporated by reference). These and other methods forsynthesizing oligionucleotides are described in Goodchild, BioconjugateChemistry 1(3):165-187 (1990), which is hereby incorporated byreference.

[0097] The nucleic acid molecules which hybridize under stringentconditions to nucleic acid molecules encoding a chemokine of the presentinvention can be used as probes in hybridization assays to detect breastdisease in a patient. For example, a sample of tissue or body fluid fromthe patient is contacted with a nucleic acid probe which, understringent conditions, hybridizes to a nucleic acid molecule encoding achemokine according to the present invention or to a complement thereof.The contacting is carried out under conditions effective to permitformation of a hybridization complex between the probe and breast tissuespecific nucleic acid molecules (i.e., the nucleic acid moleculesencoding chemokines of the present invention). Breast disease is thendetected by detecting the hybridization complex.

[0098] As used herein, the term “probe” refers to an oligonucleotidewhich forms a duplex structure with a sequence of a target nucleic acid(e.g., a nucleic acid molecule which encodes a chemokine of the presentinvention) due to complementary base pairing. The probe will contain ahybridizing region, which is a region of the oligonucleotidecorresponding to a region of the target sequence. A probeoligonucleotide either can consist entirely of the hybridizing region orcan contain additional features which allow for the detection orimmobilization of the probe but do not alter the hybridizationcharacteristics of the hybridizing region. The term “probe” also refersto a set of oligonucleotides which provide sufficient sequence variantsof the hybridization region to permit hybridization with each member ofa given set of target sequence variants. Additionally, a probe cancontain mismatches with some or all members of a given set of targetsequence variants, provided that it contains sufficient regions ofcomplementarity with each target sequence variant to permithybridization with all target sequence variants under suitableconditions.

[0099] Samples of the patient's tissue or body fluids suitable for theuse in the detection method using probes include those which arediscussed above with regard to detection methods employing antibodies.

[0100] Detection of the hybridization complex can be carried out by avariety of conventional methods. These include electrophoresis, DNAsequencing, blotting, microplate hybridization, or microscopicvisualization. Alternatively, the probe can have bound thereto a label,such as detectable functional nucleotide sequence (e.g., a T7 site, arestriction site, and the like) or one of the labels described above assuitable for use in the detection method of the present inventionemploying antibodies. Detection, in this case, involves detecting thepresence of the label, for example using the techniques discussed aboveor by using one of the conventional methods for detecting detectablefunctional nucleotide sequences.

[0101] The nucleic acid molecules which hybridize under stringentconditions to nucleic acid molecules encoding a chemokine of the presentinvention can also be used as primers in a DNA amplification assay todetect breast disease in a patient. For example, a sample of tissue orbody fluid from the patient can be contacted with a nucleic acid primerwhich, under stringent conditions, hybridizes to a nucleic acid moleculeencoding a chemokine according the present invention or to a complementthereof. The sample of tissue or body fluid from the patient in contactwith the nucleic acid primer is then treated under conditions effectiveto amplify breast tissue specific nucleic acid molecules, and the breasttissue specific nucleic acid molecules, thus amplified, are thendetected.

[0102] As used herein, the term “primer” refers to an oligonucleotide,whether natural or synthetic, capable of acting as a point of initiationof a DNA synthesis under conditions which produce a primer extensionproduct complementary to a nucleic acid strand is induced. Generally,the DNA synthesis is carried out in the presence of four differentnucleoside triphosphates and an agent for polymerization (e,g., DNApolymerase or reverse transcriptase) in an appropriate buffer (e.g.,Tris-HCl), and at suitable temperatures (e.g., at an annealingtemperature of from about 45 to about 85° C.; at an extendingtemperature of from about 55 to about 75° C.; and at a meltingtemperature of about 95° C.). The primer is preferably a single-strandedDNA. The optimal length of the primer depends on the primer's intendeduse but typically ranges from 15 to 35 nucleotides. Short primermolecules generally require cooler temperatures to form sufficientlystable hybrid complexes with the template. A primer need not complementthe exact sequence of the template but must be sufficientlycomplementary to hybridize with a template. Primers can incorporateadditional features which allow for the detection or immobilization ofthe primer but do not alter the basic property of the primer, that ofacting as a point of initiation of DNA synthesis. The term “primer”, asused herein, also refers to a set of oligonucleotides which providesufficient sequence variants of the hybridization region to permithybridization with each member of a given set of target sequencevariants, so as to act as a point of initiation of DNA synthesis.Additionally, a primer may consist of one or more oligonucleotides whichcontain mismatches with some or all members of a given set of targetsequence variants, but contains sufficient regions of complementaritywith each target sequence variant so as to enable hybridization with alltarget sequence variants under suitable conditions. The term “consensusprimers” is used herein to refer to primers containing a singleoligonucleotide complementary to a consensus target sequence, to primersconsisting of multiple oligonucleotides complementary to a consensustarget sequence, and to combinations thereof.

[0103] Samples of the patient's tissue or body fluids suitable for theuse in the detection method using probes include those which arediscussed above with regard to detection methods employing antibodies.

[0104] Amplification of breast tissue specific nucleic acid molecules(i.e., nucleic acid molecules encoding the chemokines of the presentinvention) is preferably carried out by PCR. Use of PCR to amplify DNAis described in U.S. Pat. No. 4,683,195 to Mullis et al., U.S. Pat. No.4,683,202 to Mullis, and U.S. Pat. No. 4,965,188 to Mullis et al., whichare hereby incorporated by reference. Briefly, PCR amplification of DNAinvolves repeatedly heat-denaturing the DNA, annealing twooligonucleotide primers to sequences that flank the DNA segment to beamplified, and extending the annealed primers with DNA polymerase. Theprimers hybridize to opposite strands of the target sequence and areoriented so DNA synthesis by the DNA polymerase proceeds across theregion between the primers, effectively doubling the length of that DNAsegment. Moreover, because the extension products are also complementaryto and capable of binding primers, each successive cycle essentiallydoubles the amount of DNA synthesized in the previous cycle. Thisresults in the exponential accumulation of the specific target fragmentat a rate of approximately 2^(n), where n is the number of cycles. Dueto the enormous amplification possible with the PCR process, smalllevels of DNA carryover from samples with high DNA levels can result inPCR product, even in the absence of purposefully added template DNA.Optimally, all reaction mixes are set up in an area separate from PCRproduct analysis and sample preparation and care is taken to avoid crosscontamination, for example, by using dedicated or disposable vessels,solutions, pipettes (preferably positive displacement pipettes), andpipette tips (preferably with aerosol barriers) for RNA/DNA, reactionmixing, and sample analysis. See e.g., Higuchi et al., Nature339:237-238 (1989) and Kwok et al. in Innis et al., eds., PCR Protocols:A Guide to Methods and Applications, San Diego, Calif.: Academic Press,Inc., pp. 142-145 (1990), which are incorporated herein by reference.

[0105] Primers suitable for use in the method of the present inventionare preferably 15 to 30 nucleotides in length and are designed to have ahigh degree of homology with breast tissue specific nucleic acidsequences (i.e., with nucleic acid molecules encoding chemokines of thepresent invention). For each region to be amplified, two regions ofhomology are required, one for negative-strand primers and another forpositive-strand primers. Once a homologous region is identified, aconsensus primer is designed. Degenerate bases can be used in the designto accommodate positions at which an individual breast tissue genevaries in sequence from the consensus sequence (genetic polymorhpism).Preferably, as many degenerate positions are made as is necessary sothat all breast tissue sequences have fewer than three mismatches withthe consensus primer. Any mismatches that are not accommodated by thedegenerate positions in the primer should preferably be located morethan 3 bases from the 3′ end of the primer. Likewise, any degeneratepositions should preferably be more than 3 bases from the 3′ end of theprimer. Degenerate primers having estimated minimum and maximum Tms ofabout 54° C. and about 64° C., respectively, are preferred, where Tmsare estimated by summing a contribution from each base pair. In thisformulation, each G or C contributes 4° C. to the Tm, and each A or Tcontributes 2° C. to the Tm. Finally, it is generally preferred thatprimers be designed so that they do not span palindromes or repetitivesequences.

[0106] Following amplification, the breast tissue specific nucleic acidmolecules are detected to determine whether amplification has occurred.Since amplification will occur (and breast tissue specific nucleic acidmolecules will be detected) only if some amount of breast tissuespecific nucleic acid molecules were present in the sample beforeamplification, detection of breast tissue specific nucleic acidmolecules after amplification indicates the presence of breast diseasein the patient from which the sample came. Suitable nucleic acid primersinclude those which, under stringent hybridization conditions, hybridizeto a nucleic acid molecule encoding a chemokine having an amino acidsequence corresponding to SEQ ID NO:1 and/or which hybridize to anucleic acid molecule having a nucleotide sequence corresponding to SEQID NOs:. 6-8. In particular, suitable nucleic acid primers include thosehaving a nucleotide sequence corresponding to SEQ ID NO:9 or SEQ IDNO:10.

[0107] There are a variety of known methods for determining whetheramplification has occurred. For example, a portion of the PCR reactionmixture can be subjected to gel electrophoresis, the resulting gel canbe stained with, for example, a ultraviolet absorbing stain, such aswith ethidium bromide, and the stained gel can be exposed to ultravioletlight to determine whether a product of the expected size can beobserved. Alternatively, labeled PCR primers or labeleddeoxyribonucleoside 5′-triphosphates can be used to incorporation thelabel into the amplified DNA. The presence of a breast tissue specificnucleic acid amplification product can then be detected by detecting thelabel. Examples of suitable labels and label detection methods includethose set forth above with regard to the detection method which employedhybridization. Another method for determining if amplification hasoccurred involves testing a portion of the amplified reaction mixturefor ability to hybridize to a labeled probe designed to hybridize onlyto the amplified DNA. Amplified breast tissue specific nucleic acidmolecules can also be detected by DNA sequencing as well as bymicroscopic visualization.

[0108] A number of treatments can be used to amplify the breast tissuespecific nucleic acid molecules (i.e., nucleic acid molecules encoding achemokine of the present invention). These include PCR, ligase chainreaction (“LCR”), self-sustained sequence (“3 SR”) replication, Q-betareplicase, nucleic acid sequence based amplification (“NASBA”),transcription-based amplification System (“TAS”), or branched-DNAmethods.

[0109] Although PCR is the preferred amplification method, amplificationof target sequences in a sample may be accomplished by any knownamplification method, such as ligase chain reaction methods (described,e.g., in Wu et al., Genomics 4:560-569 (1988), which is herebyincorporated by reference). In LCR, the consensus primers can be used todirect the joining of oligonucleotide segments that anneal to the targetnucleic acid, thereby amplifying the target. Further details with regardto this method can be found in, for example, WO 89/09835, which ishereby incorporated by reference. Other suitable amplification methodsinclude the TAS amplification system (described, e.g., in Kwoh et al.,Proc. Natl. Acad. Sci. USA 86:1173-1177 (1989), which is herebyincorporated by reference), branched-DNA methods (described, e.g., inKern et al., J. Clin. Microbiol. 34:3196-3202 (1996), which is herebyincorporated by reference), and self-sustained sequence replicationmethods (described, e.g., in Guatelli et al., Proc. Natl. Acad. Sci. USA87:1874-1878 (1990), which is hereby incorporated by reference). Each ofthese methods provides sufficient amplification so that the targetsequence can be detected by nucleic acid hybridization to anoligonucleotide probe, such as those described above, or by otherdetection methods. Alternatively, methods that amplify the probe todetectable levels, such as Q-beta replicase amplification can beemployed. This method is described in, for example, Kramer et al.,Nature 339:401-402 (1989) and Lomeli et al., Clin. Chem. 35:1826-1831(1989), which are hereby incorporated by reference. Further detailsregarding these and other suitable amplification methods are provided inAbramson et al., Current Opinion in Biotechnology 4:41-47 (1993), whichis hereby incorporated by reference. The term “probe”, as used withregard to the above amplification methods, encompasses any of thesequence-specific oligonucleotides used in these procedures. Forinstance, the two or more oligonucleotides used in LCR are “probes” forpurposes of the present invention, even though some embodiments of LCRonly require ligation of the probes to indicate the presence of anallele.

[0110] In some cases, the tissue or fluid sample from the patient maycontain a breast tissue specific nucleic acid transcript (i.e., mRNA)which codes for the chemokine of the present invention. In thissituation, the mRNA can be converted to cDNA by reversetranscription-PCR (“RT-PCR”) prior to amplification. This involvestreating the mRNA-containing sample with reverse transcriptase in anappropriate reaction mixture and in the presence of an appropriateprimer. The primer used in the reverse transcription reaction can be aconsensus primer of the present invention, or it can be a differentoligonucleotide that hybridizes near the 3′ end of the mRNA. Althoughrandom hexamers are not specific for the 3′ end of the mRNA molecule,they are suitable for reverse transcription of mRNA to provide a cDNAtemplate for amplifying breast tissue specific nucleic acids. This cDNAcopy is then made into a double stranded DNA molecule, which can beamplified as described above.

[0111] The nucleic acid primer used in the above amplification detectionmethod may be assembled as a kit for detecting breast disease. Such akit includes consensus primers and molecular probes. A preferred kitalso includes the components necessary to determine if amplification hasoccurred. The kit may also include, for example, PCR buffers andenzymes; positive control human breast tissue specific sequences,reaction control primers, such as betaglobin primers; and instructionsfor amplifying and detecting breast tissue specific sequences.

[0112] The symbols used herein to designate particular nucleotides areset forth below in Table 2. TABLE 2 Symbol Meaning G guanine A adenine Tthymine C cytosine R adenine or guanine Y cytosine or thymine M adenineor cytosine K guanine or thymine S cytosine or guanine W adenine orthymine H adenine or cytosine or thymine B cytosine or guanine orthymine V adenine or cytosine or guanine D adenine or guanine or thymineN adenine or cytosine or guanine or thymine

EXAMPLES Example 1 Isolation of Novel Human Breast Tissue SpecificNucleic Acid Sequences Using Suppression Subtractive Hybridization

[0113] Suppression Subtractive Hybridization (“SSH”) was performedaccording to the protocol of Diatchenko et al., Proc. Natl. Acad. Sci.USA 93:6025-6030 (1996), which is hereby incorporated by reference,using commercial reagents from Clontech (PCR-Select cDNA subtractionkit). Human polyA RNAs derived from bone marrow, skeletal muscle, lung,liver, pancreas, and mammary gland were obtained from Clontech, and 2 mgof each were reverse transcribed. The cDNAs derived from mammary glandwere subdivided and ligated to different cDNA adaptors according to themanufacturer's protocol. Primary and secondary subtractivehybridizations were performed by adding an excess of denatured cDNAsderived from human bone marrow, lung, pancreas, liver, and skeletalmuscle (“driver” cDNAs”) to the mammary gland cDNA (“tester cDNA”). Theentire population of subtracted molecules was subjected to two rounds ofDNA amplification: a primary PCR to amplify differentially expressedsequences and a secondary (nested) PCR to enrich for those sequences.PCR primers 1 and 2 and nested PCR primers 1 and 2 (Clontech) were usedin accordance with the protocol of the PCR-Select cDNA subtraction kitfor primary and secondary PCR, respectively. All DNA amplifications wereperformed with a Perkin-Elmer DNA Thermal Cycler Model 2400 usingparameters of 94° C., 5 seconds (denature); 68° C., 30 seconds (anneal);and 72° C., 150 seconds (extend) and using the Advantage KlentaqPolymerase Mix (Clontech) which contains a TaqStart Antibody to provideautomatic hot start PCR (Kellogg et al., Biotechniques 16:1134-1137(1994), which is hereby incorporated by reference). PCR was optimizedusing the control reagents contained in the PCR-Select cDNA subtractionkit as template and the OPTI-PRIME PCR Optimization Kit (Stratagene).Amplification products were analyzed by gel electrophoresis (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor, N.Y: Cold Spring Harbor Laboratory Press (1987) (“Sambrook”) andAusubel et al., Current Protocols in Molecular Biology, New York: GreenePublishing Associates and Wiley-Interscience (1990) (“Ausubel”), whichare hereby incorporated by reference). In our hands, the optimal bufferfor primary PCR contained 40 mM Tricine-KOH (pH 9.2), 15 mM KOAc, 3.5 mMMg(OAc)₂, and 75 mg/ml bovine serum albumin (10× Klentaq PCR reactionbuffer, Clontech). The optimal buffer for secondary PCR contained 10 mMTris-HCl (pH 8.3), 75 mM KCl, and 3.5 mM MgCl₂ (Stratagene, Opti-Prime1× Buffer #4) with 5% dimethylsulfoxide.

Example 2 Cloning of the Subtracted cDNAs

[0114] Nested PCR primer 1 was phosphorylated using reagents fromInvitrogen (Eukaryotic TA Cloning Kit, Unidirectional). Secondary PCR(10 cycles) was performed in the optimized buffer described above usingnested PCR primer 2 and the phosphorylated nested PCR primer 1. PCRproducts were directionally ligated into the mammalian expression TAcloning vector pCR™3.1-Uni and transformed into TOP10F′ competent cellsusing general techniques (Sambrook and Ausubel, which are herebyincorporated by reference) and commercial reagents from InVitrogen.PCR™3.1-Uni contains a T-overhang which allows the direct cloning of PCRproducts containing single 3′ A-overhangs (Mead et al., Bio/Technology9:657-663 (1991), which is hereby incorporated by reference. Transformedcells were selected in Luria-Broth media containing 25 mg/ml kanamycin.

Example 3 Sequencing of Differentially Expressed Clones

[0115] DNA plasmid isolations were performed using the Qiagen PlasmidMini Kit which employs the alkaline lysis method (Sambrook, which ishereby incorporated by reference). Plasmids were screened for insertsequences using nested PCR primers 1 and 2 and the protocol and reagentsfrom the Geneamp PCR Kit (Perkin Elmer), and amplified products wereanalyzed by gel electrophoresis. Clones containing inserts greater than100 basepairs (“bp”) were obtained for sequencing analysis. Dideoxy DNAsequencing was performed using the Applied Biosystems Model 373Automated DNA Sequencing System. The DNA sequence of each strand wasdetermined using sequencing primers T7 (5′ TAATACGACTCACTATAGGG 3′) (SEQID NO: 12) and pCR™3.1 Reverse (5′ TAGAAGGCACAGTCGAGG 3′) (SEQ IDNO:13), respectively.

Example 4 Search for Genetic Homologies

[0116] GenBank was searched for homologous sequences via the programBLASTN (Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Benson etal., Nucleic Acids Res. 24:1-5 (1996), which are hereby incorporated byreference). Sequences were classified as known or unknown based on theresulting score and probability values. Known sequences were arbitrarilydefined as those having probability values greater than 0.05 (p>0.05)relative to database sequences or those showing homology to non-humanspecies or to cosmids containing human DNA of which a function has notbeen assigned.

Example 5 Rapid Amplification of cDNA Ends

[0117] Full length mammary associated chemokine (“MACK”) cDNA wasgenerated using 5′ and 3′ rapid amplification of cDNA ends (“RACE”)(Frohman, PCR Protocols, New York: Academic Press, pp. 28-39 (1990),which is hereby incorporated by reference) using commercial reagents(Marathon cDNA Amplification Kit, Clontech). Human mammary gland polyARNA (Clontech) was used as a template for first and second strand cDNAsynthesis, and adaptors were ligated to the pool of cDNA according tothe manufacturer's protocol. The 3′ RACE product was obtained by usingthe gene-specific primer (24R) 5′ ACTGGGATTATAGGTGTGAGCC 3′ (SEQ ID NO:14) and Clontech's adaptor primer 1 (AP1) using “Touchdown PCR”according to the manufacturer's directions. This was followed by asecondary PCR using the nested gene-specific primer (24R2) 5′CAAATTCAGGTTTAATGTTTTTGG 3′ (SEQ ID NO:15) and Clontech's nested adaptorprimer 2 (AP2). PCR products were cloned into the T/A cloning vectorpCR2.1 (Invitrogen). DNA plasmid preparations were prepared andsequenced using vector sequences T7 and Ml 3 reverse. Internalsequencing primers were based on confirmed sequences.

[0118] The 5′ RACE product was obtained using Clontech's MARATHON™ ReadycDNA from human mammary gland according to their protocol. “TouchdownPCR” was performed on the cDNA using gene-specific primer (F4) 5′CTCAAACGTGTGAGCCCGGCA 3′ (SEQ ID NO:16) and AP1, and nested PCR wasperformed using nested gene-specific primer (F3) 5′GCTACTCAAACTAGACGTTTTGAAG 3′ (SEQ ID NO:17) or (F1) 5′GAATTCACGTAGGAAATTCTTAACC 3′ (SEQ ID NO:9) and AP2 (see above). PCRproducts were cloned and sequenced as described above. A consensussequence was generated using programs from the Hitachi software packageDNAsis for Windows.

Example 6 Northern Blot Analysis

[0119] Human mammary gland PolyA+RNA (3 μg, Clontech Laboratories, Inc.)was separated and transferred using the NORTHERNMAX™ Northern BlottingKit from Ambion. PCR amplification of a 302 bp region within thepredicted ORF was performed using primers F8 5′ CCGTATGTTTCGTGTTTCCCCTGA3′ (SEQ ID NO: 18) and R5 5′ AGCCATACTTCCCATTGCCTCCAG 3′ (SEQ ID NO: 19)and 5′ RACE clone (#27) as template. This fragment was directionallyligated to a T7 promoter (LIG'NSCRIBE™ RNA Polymerase Promoter AdditionKit, Ambion) and amplified such that the antisense strand was orientatedimmediately downstream to the T7 promoter according to themanufacturer's protocol. An antisense riboprobe having SEQ ID NO:11 wastranscribed in vitro using T7 RNA polymerase, and labeled using theBRIGHTSTAR™ Psoralen-Biotin Nonisotopic Labeling Kit (Ambion).Hybridization and chemiluminescent detection were performed usingprotocols from Ambion's NORTHERNMAX™ and BRIGHTSTAR™ BIODETECT™ Kits,respectively.

Example 7 Production of Antisera to the Open Reading Frame ProteinSequence

[0120] The predicted open reading frame within the MACK gene wasdetermined using commercial software (DNAsis, Hitahci Corp.). Syntheticpeptides corresponding to predicted immunogenic domains, KLH-peptideconjugates and resultant rabbit antisera were produced by ResearchGenetics, Inc. (Huntsville, Ala.). Antisera were collected after a10-week immunization protocol.

Example 8 Titration of Anti-peptide Antisera

[0121] Synthetic peptides were dissolved in 0.2 M carbonate-bicarbonatebuffer, pH 9.4 (CBC buffer) at a concentration of 10 μg/mL. Microplateswere coated (100 (μL/well) with the peptides at 4° C. for 18 hrs. Thesolution was removed and the microwells were blocked with 1% bovineserum albumin in tris-buffered saline (“TBS”), pH 7.4 for 1 hr.Dilutions of anti-peptide antisera were incubated with the solid-phasepeptides for 1 hr, and, following a wash procedure, goat antibodies torabbit immunoglobulin (biotin-conjugated) were added for 30 min. Afteranother wash procedure, each well received 100 μL of avidin-biotinylatedalkaline phosphatase complex (ABC Kit, Pierce Immunochemicals) for 30min. Thereafter, the wells were washed, and substrate (para-nitrophenylphosphate, 1 mg/ml in diethanolamine buffer, pH 9.8) was added for 30min. After stopping the reactions with 50 μL of 5 N NaOH, opticaldensity was determined at an absorbance of 450 nm using a microplatespectophotometer.

Example 9 Purification of IgG and Enzyme Coupling

[0122] IgG from rabbit serum was purified using protein A affinitychromatography (MAPS II Kit, Bio-Rad Labs). IgG was conjugated tohorseradish peroxidase using the periodate oxidation technique (Nakaneet al., J. Histochem. Cytochem. 22:1084-1091 (1974), which is herebyincorporated by reference).

Example 10 SDS-PAGE and Western Blotting

[0123] SDS-PAGE was performed as described in Laemmli, Nature227:680-685 (1970) (“Laemmli”), which is hereby incorporated byreference.

[0124] Western blotting was performed essentially as described inPapsidero et al., Hybridoma 7:117-128 (1988), which is herebyincorporated by reference, using nitrocellulose paper with a 0.22 μmpore size. Blots were incubated for 1 hr at room temperature with immuneor pre-immune sera diluted in assay buffer. The membranes were washedand developed with avidin-biotin-alkaline phosphatase reagents usingcommercial reagents (ABC Kit, Pierce Immunochemicals). Blots weredeveloped with insoluble substrate (BCIP/NBT solution, PierceImmunochemicals), washed in water and air-dried.

Example 11 Results of Comparison of Isolated Sequence Tags to GenBank

[0125] Human breast tissue mRNA was subjected to SSH and 118 sequencetags were isolated and sequenced. Of the total examined, 62% (73 of 118)were homologous to genes found in the GenBank database (Table 3). Ofinterest, approximately 14% (10 of 73) of the previously describedsequences were breast tissue specific or highly associated with breasttissue (i.e., casein isoforms, alpha-lactalbumin, and milk fat globuleproteins). Remarkably, 38% of the sequence tags (45 of 118) demonstratedno significant homology with genes found in the database (Table 3).These novel genes were studied further using RT-PCR in order todetermine the specificity of their tissue expression. TABLE 3 HumanBreast Tissue mRNA Sequence Tags Isolated Using Suppression SubtractionHybridization insert Identical ID GenBank size Blast Scoreresidues/Total # Search (bp) Strongest Homology (probability) residues(%) 1 Known 309 Human keratin 459 (p < 0.001) 99/108 (91%) 5 Known 195Human A1S9 mRNA 619 (p < 0.001) 127/133 (95%) 7 Known 66 Human Vimentin330 (p < 0.001) 66/66 (100%) 8 Unknown 198 S. cerevisiae 114 (p = 1.0)30/39 (76%) 10 Known 96 H. sapiens rho GAP protein 462 (p < 0.001) 95/98(96%) 11 Known 105 Mouse cerbA alpha 2 mRNA 507 (p < 0.001) 105/105(100%) (thyroid H.) 14 Known 135 TCR eta = Tcell receptor eta chain 258(p < 0.001) 62/75 (82%) 16 Known 115 Pancreatic peptidylglycine 557 (p <0.001) 113/115 (98%) 20 Known 182 H. sapiens paraoxynase 520 (p < 0.001)122/146 (83%) 22 Known 194 Human mRNA for cytoskeletal 956 (p < 0.001)192/194 (99%) gamma actin 23 Unknown 201 Chimpanzee cmyc protooncogene134 (p = 0.18) 42/61 (68%) 28 Known 150 Milk fat globule protein (human)515 (p < 0.001) 103/103 (100%) 30 Known 442 H. sapiens mitochondrialgenome 1245 (p < 0.001) 251/254 (98%) 47 & Unknown 143 Beet necroticyellow vein virus 134 (p = 0.10) 54/88 (61%) 67 51 Known 174 H. sapiensmRNA homologue to yeast 831 (p < 0.001) 169/174 (97%) ribo. Protein 54Unknown 125 M. musculus for Notch 3 179 (p < 0.001) 45/57 (78%) 57 Known180 H. sapiens cDNA for betacasein 715 (p < 0.001) 147/155 (94%) 60Unknown 202 X. laevis mRNA for DNA binding 122 (p = 0.88) 42/64 (65%) 61Known 286 Human 28 k basic protein 1349 (p < 0.001) 273/278 (98%) 62Known 195 Human A1S9 mRNA 968 (p < 0.001) 194/195 (99%) 74 Known 152Human MER 37 transposable element 351 (p < 0.001) 87/108 (80%) 75 Known192 Human mRNA for cytoskeletal 960 (p < 0.001) 192/192 (100%) gammaactin 78 Unknown 626 C. elegans ZK1073 123 (p = 1.0) 31/39 (79%) 79 &Unknown 90 & 100 Myxococcus xanthus photolyase 113 (p = 0.96) 29/37(78%) 80 82 Unknown 295 Actinobacillus riboflavin biosynthesis 121 (p =0.99) 41/62 (66%) operon 89 Known 214 Human casK mRNA for Kappa casein1063 (p < 0.001) 213/214 (99%) 101 Unknown 99 C. elegans C35B8 118 (p =0.71) 34/47 (72%) 105 Known 84 H. sapiens mRNA for 90K product 357 (p <0.001) 75/84 (89%) 114 Unknown 111 Human peregrin mRNA 127(p = 0.23)39/56 (69%) 115 Known 186 Rat 8s RNA 728 (p < 0.001) 147/151 (97%) 116Unknown 413 M. musculus for p38264 787 (p < 0.001) 171/190 (99%) 120Known 253 Human SF 2 p33 mRNA (splicing 1223 (p < 0.001) 247/253 (97%)factor) 121 Unknown 154 M. musculus serum inducible 653 (p < 0.001)141/154 (91%) 122 Unknown 354 Drosophila silver p. 264 (p < 0.001)118/202 (58%) 127 Known 133 H. sapiens mRNA for rat HREV 368 (p < 0.001)96/125 (76%) 107 like 131 Unknown 117 C. elegans R12C12 126 (p = 0.31)38/54 (70%) 133 Unknown 133 Bos taurus polymeric immunoglobulin 149 (p <0.001) 33/37 (89%) 135 Unknown 124 Rat vesicle associated membrane 286(p < 0.001) 60/64 (93%) protein 140 Known 312 Human ferritin 1530 (p <0.001) 308/312 (98%) 142 Unknown 123 Human MAGE 4a antigen gene 129 (p =0.21) 37/51 (72%) 143 Known 94 Human ribosomal protein L28 470 (p <0.001) 94/94 (100%) 145 Unknown 283 M. auratus beta myosin 132 (p =0.39) 52/84 (61%) 152 Known 551 H. sapiens mitochondrial genome 751 (p <0.001) 153/157 (97%) 155 Unknown 238 R. norvecigus adenylyl cyclase 109(p = 0.87) 35/52 (67%) 158 Known 186 Rat 8s RNA 698 (p < 0.001) 142/146(97%) 162 Known 129 Gamma actin 629 (p < 0.001) 127/129 (98%) 164 Known95 Human mRNA for OSFI 452 (p < 0.001) 92/95 (96%) 171 Known 321 HumanmRNA for cytokeratin 1033 (p < 0.001) 209/213 (98%) 175 Unknown 134 M.musculus isocitrate dehydrogenase 130 (p = 0.19) 36/49 (73%) 176 Known150 Human mitochondrial DNA 750 (p < 0.001) 150/150 (100%) 178 Unknown269 Gorilla ALU repeat/H. sapiens casein 191 (p < 0.001) 47/60 (78%)kinase 179 Known 182 Human COREI protein 903 (p < 0.001) 181/182 (99%)181 Known 155 Human alphalactalbumin 712 (p < 0.001) 144/147 (97%) 182 &Unknown 259 Human DNA sequence from cosmid 196 (p < 0.001) 78/127 (61%)197 N28H9 188 Known 216 Human ALU 453 (p < 0.001) 101/114 (88%) 189Unknown 105 Human DNA sequence from cosmid 125 (p < 0.001) 31/39 (79%)N37F 192 Unknown 104 M. musculus cytoplasmic protein 119 (p = 0.62)27/31 (87%) 195 Known 155 Human alphalactalbumin 696 (p < 0.001) 144/147(97%) 196 Known 156 Mouse 28s rRNA 412 (p < 0.001) 84/86 (97%) 201 Known183 Human COREI protein 841 (p < 0.001) 169/171 (98%) 204 Unknown 194Human DNA sequence from cosmid 514 (p < 0.001) 118/138 (85%) L139H 205Known 54 Human cytokeratin 238 (p < 0.001) 48/49 (97%) 207 Known 139Human prostasin 589 (p < 0.001) 119/121 (98%) 208 Unknown 356 Humancathepsin D (catD) gene 130 (p = 0.64) 34/44 (75%) 209 Known 373Putative zinc finger Rattus norxecigus 707 (p < 0.001) 161/195 (82%) 210Known 129 Gamma actin 606 (p < 0.001) 124/129 (97%) 214 Known 105Alphalactalbumin 509 (p < 0.001) 103/105 (98%) 216 Known 153Alphalactalbumin 709 (p < 0.001) 143/145 (98%) 218 Known 190 Acidiccalponin 941 (p < 0.001) 189/190 (99%) 220 Unknown 99 C. elegans cosmidC34E7 108 (p = 1.0) 28/36 (77%) 221 Unknown 122 S. cerevisiae chromosome121 (p = 0.33) 22/25 (87%) 223 Unknown 91 Bovine betahydroxylase 113 (p= 0.94) 29/37 (78%) 224 Known 164 Lactate dehydrogenase 614 (p < 0.001)124/127 (97%) 225 Known 273 Proalpha collagen 1335 (p < 0.001) 269/273(98%) 229 Known 235 Collagen 1143 (p < 0.001) 232/235 (98%) 230 Unknown117 Plasmodium falciparum (strain FCR3) 116 (p = 0.89) 30/39 (76%) 231 &Unknown 94 CNS myelin P0like glycoprotein 124 (p = 0.26) 40/59 (67%) 234232 Unknown 405 H. sapiens mRNA for 218kD Mi2 132 (p = 0.55) 42/62 (67%)protein 233 Unknown 198 Rat TnT gene encoding troponin T 130 (p = 0.36)34/44 (77%) 238 Known 140 Human Thy 1 glycoprotein 645 (p < 0.001)133/140 (95%) 242 Known 136 H. sapiens casK mRNA for Kappa 666 (p <0.001) 134/136 (98%) casein 249 Known 136 H. sapiens casK mRNA for Kappa680 (p < 0.001) 136/136 (100%) casein 250 Known 288 H. sapiens CpG DNA792 (p < 0.001) 164/172 (95%) 252 Known 525 Human pHL1 gene (cmyconcogene) 1704 (p < 0.001) 352/377 (93%) 253 Known 125 Human mRNA forplasma gelsolin 618 (p < 0.001) 124/125 (99%) 255 Known 138 Human Xq 28genomic DNA 333 (p < 0.001) 69/74 (93%) 256 Known 56 Human vimentin 280(p < 0.001) 55/55 (100%) 257 Known 236 Human breast cancer LIV1regulated 1134 (p < 0.001) 230/236 (97%) mRNA 258 Known 125 Humangelsolin 618 (p < 0.001) 124/125 (99%) 261 Known 283 Human mRNA for ORFmyeloblast 1394 (p < 0.001) 280/283 (98%) celline 263 Known 156 Humanphemphigoid autoantigen 773 (p < 0.001) 155/156 (99%) 264 Unknown 198 C.elegans N2 basichelix 116 (p = 0.99) 36/52 (69%) 269 Unknown No MatchesIdentified N/A N/A 275 Known 283 Human mRNA for ORF 1373 (p < 0.001)277/283 (97%) 276 Unknown 195 C. elegans cosmid ZK813 133 (p = 0.20)41/59 (69%) 279 Known 339 Alpha casein 1674 (p < 0.001) 336/339 (99%)284 Known 129 H. sapiens BTF2p44 mRNA for basic 645 (p < 0.001) 129/129(100%) transcription 287 Known 293 Human mRNA 1251 (p < 0.001) 261/280(93%) 291 Unknown 171 D. melanogaster chromosome 3 locus 133 (p = 0.18)33/41 (80%) 85D 292 Known 148 H. sapiens HIV1 TAR RNA binding 699 (p <0.001) 143/148 (96%) protein 297 Known 136 Human migration inhibitoryfactor 617 (p < 0.001) 127/136 (93%) mRNA 300 Unknown 176 R. norvegicusFSHregulated protein 427 (p < 0.001) 91/98 (92%) mRNA 302 Unknown 96 S.platensis rpsB gene (ribosomal 111 (p = 0.99) 43/69 (62%) protein S2)303 Known 146 H. sapiens alphalactalbumin 705 (p < 0.001) 141/141 (100%)305 Known 99 B. taurus myosin IB mRNA 336 (p < 0.001) 80/99 (80%) 308Unknown 295 D. melanogaster Oregon R mRNA 422 (p < 0.001) 134/197 (68%)314 Unknown 158 Maize mRNA for catalase 2 113 (p = 1.0) 29/37 (78%) 329Unknown 160 C. elegans cosmid C09B9 117 (p = 0.97) 39/59 (66%) 330 Known109 Human nonmuscle myosin alkali light 531 (p < 0.001) 107/109 (98%)chain 333 Unknown 119 Mouse MA3 (apoptosisrelated gene) 124 (p = 0.39)30/37 (81%) mRNA 337 Unknown 99 No Matches Identified N/A N/A 338Unknown 271 Human fur gene, exons 1 through 8 143 (p = 0.057) 51/79(64%) 339 Known 65 H. sapiens mRNA for IgG1 heavy 123 (p = 0.012) 35/48(72%) chain

[0126] At least one expressed sequence tag (Table 3, ID # 189),designated Breast Sequence Tag-24 (BRST-24″), was demonstrated toexhibit a high level of specificity to breast tissue. BRST-24 has SEQ IDNO:35 as follows: ACACGAATTCACGTAGGAAATTCTTAACCAAAAACATTAAACCTGAATTTGATCACAAGAAAATAATTAGGCCAGGCACTGTGGCTCACACCTATAATCC CAGT

Example 12 Tissue Specificity Analysis of BRST-24 Using RT-PCR

[0127] The tissue specificity of BRST-24 was experimentally demonstratedusing RT-PCR analysis of various human tissue mRNAs along with primerswhich are complementary to regions of the BRST-24 nucleotide sequence.The primers had the following sequences: GAATTCACGTAGGAAATTCTTAACC (F1primer) ACTGGGATTATAGGTGTGAGCC (R1 primer)

[0128] These sequences are respectively identified herein as SEQ ID NO:9and SEQ ID NO:10.

Example 13 Detection of BRST-24 Using RT-PCR Analysis of Human Tissues

[0129] RT-PCR was performed using the protocol and reagents from thePerkin-Elmer GeneAmp EZ rTth RNA PCR Kit. PCR primers BRST-24 fwd (5′GAATTCACGTAGGAAAT TCTTAACC 3′) (SEQ ID NO:9) and BRST-24 rev (5′ACTGGGATTATAGGTGTGAGCC 3′) (SEQ ID NO: 10) were synthesized by ResearchGenetics. A tissue panel of total RNAs derived from human testis, brain,lung, prostate, kidney, skeletal muscle, small intestine, liver,pancreas, uterus, and breast (all obtained from Clontech) was screenedvia RT-PCR for the presence of BRST-24 using a Perkin-Elmer DNA ThermalCycler Model 2400. Reverse transcription was carried out for 30 minutesat 60° C., the reaction mix was denatured at 94° C. for one minutefollowed by 40 cycles of PCR (94° C., 15 seconds (denature), 60° C., 30seconds (anneal and extend)), and a final extension was carried out for7.0 minutes at 60° C. The amplified products were observed on a 3%agarose gel (0.5× TBE) as described in Sambrook, which is herebyincorporated by reference.

[0130] As shown in Table 4, the BRST-24 primer pair was able to beutilized to amplify nucleotide sequences from all of three specimens ofhuman breast tissue mRNA using RT-PCR. These specimens included twonormal breast tissue pools and one specimen of invasive ductalcarcinoma. Other human tissue mRNAs examined were noted to contain nodetectable, amplifiable mRNA genetic sequences corresponding to BRST-24.These tissues included liver, lung, small intestine, pancreas, uterus,brain, kidney, and skeletal muscle. A testes specimen did, however,produce a faint reaction product. As an experimental control, mRNAsequences specific for prostate specific antigen (“PSA”) were detectedby RT-PCR using primers homologous to regions within the PSA nucleicacid sequence (Deguchi et al., Cancer Research 53:5350-5354 (1993),which is hereby incorporated by reference). As seen in Table 4, PSA mRNAwas exclusively detected in human prostate tissue, confirming thespecificity of the PSA mRNA expression and the integrity of theexperimental protocol. TABLE 4 Differential Expression of BRST-24 andPSA Transcripts in Human Tissues as Detected Using RT-PCR Normal/BRST-24⁴ PSA⁵ Tissue Malignant Expression Expression Breast¹ Normal  ²⁺⁶ ND⁷ Breast² Normal 2+ − Breast³ Carcinoma 2+ ND Prostate Normal − 2+Kidney Normal − − Pancreas Normal − − Small Intestine Normal − −Skeletal Muscle Normal − − Testis Normal +/− − Brain Normal − − UterusNormal − − Liver Normal − − Pancreas Normal − −

[0131] Expression of BR-24 transcript was also monitored using Northernblotting with an internal probe from the BR-24 cDNA sequence having asequence corresponding to S SEQ ID NO:11.

[0132] Northern blot analysis of polyA RNA from human mammary glandresulted in the detection of a transcript appearing slightly above the3000 base pair marker. This is consistent with the predicted transcriptsize based upon results from RACE construction of the full-length cDNA.

[0133] BR-24 nucleic acid sequences were also detected in human celllines using RT-PCR along with the same primers used in the aboveexperiments. Results as, seen in Table 5, provide additional support tothe view that the BR-24 gene is expressed preferentially in humanmammary cells. TABLE 5 Detection of BR-24 Transcripts in Cultured HumanCell Lines Expression of Cell Line Description BR-24 Transcripts BT-20Breast Carcinoma 2+ MCF-7 Breast Carcinoma 1+ MDA-MB-157 BreastCarcinoma − SK-OV-3 Ovary Carcinoma − LNCaP Prostate Carcinoma − SW620Colon Carcinoma 1+−

Example 14 Isolation of the Full-Length BR-24 cDNA

[0134] To obtain the full-length cDNA sequence of MACK, the 5′ and 3′RACE clones were overlapped. Thus, this sequence represents theconsensus of 5′ and 3′ RACE clones from a population of donor mRNAs. The5′ RACE clones varied in length at the 5′ end which may be attributed tosecondary structure and pausing of the reverse transcription during cDNAsynthesis. Using this method, a consensus cDNA sequence of 3117 basepairs, excluding the polyA tail was generated. This sequence isidentified herein as SEQ ID NO:6.

[0135] Using computer algorithms (DNASis software package, HitachiCorp.), the open reading frame was determined to encode a protein of 127amino acids, between nucleic acid bases 47 and 428 above. The amino acidsequence of this protein is identified herein as SEQ ID NO:1. Thededuced molecular weight of the protein was 14,232 daltons, and thededuced isoionic point was pH 10.44.

[0136] Of interest, the above protein sequence shared sequence homologywith a class of cytokines designated as “chemokines” (See Baggiolini etal., Ann. Rev. Immunol. 15:675-705 (1997) and Rollins, Blood 90:909-928(1997), which are hereby incorporated by reference. Thus, the abovesequence represents a new member of the “CC” or “β” class of chemokines.FIG. 1 shows alignment of the MACK amino acid sequence with othermembers of the CC chemokine family. Of significance, the identificationof cytokines in human milk is of great interest and is a topic which hasbeen recently investigated (Srivastava et al., Res. Commun. Molec. Path.Pharm. 93:263-283 (1996), which is hereby incorporated by reference).

Example 15 Specificity of Anti-Peptide Antisera

[0137] Rabbit antisera were raised against three regions (underlinedtype) of the MACK protein sequence (SEQ ID NO:1): MQQRGLAIVA LAVCAALHASEAILPIASSC CTEVSHHISR RLLERVNMCR IQRADGDCDL AAVILHVKRX RICVSPHNHTVKQWMKVQAA XKNGKGNVCH RKKHHGKRNS NRAHQGKHET YGHKTPY

[0138] The sequence corresponding to amino acids 32-49 of the MACKprotein was designated “MACK A” and has an amino acid sequencecorresponding to SEQ ID NO:3. The sequence corresponding to amino acids92-107 of the MACK protein was designated “MACK B” and has an amino acidsequence corresponding to SEQ ID NO:4. The sequence corresponding toamino acids 109-127 of the MACK protein was designated “MACK C” and hasan amino acid sequence corresponding to SEQ ID NO:5.

[0139] Antisera against their respective peptides demonstrated hightiter, up to dilutions of over 100,000. In addition, anti-peptideantisera reacted with a high degree of specificity to theircorresponding immunogen.

[0140] To determine if antisera raised against peptides from the deducedprotein sequence of the MACK protein recognized the native protein,Western blotting experiments were performed. Inasmuch as the prostatetissue specific protein PSA is found in the secretion of the prostategland (i.e., seminal fluid), it was suspected that the MACK proteinwould be detectable in the secretion of the mammary gland. Of interest,when samples of human milk were examined on Western blotting versus theanti-MACK peptide antisera, each of 6 specimens was noted to contain animmunoreactive protein of having an experimentally determined weight ofapproximately 16-17 kDa. This band was not present when control blotswere allowed to react with non-immune rabbit sera, suggestingspecificity associated with the use of the anti-MACK peptide antisera.This specificity was confirmed using absorption experiments with solublepeptides. Following absorption of the anti-sera with soluble peptides(100 μg per ml of antiserum dilution), the specific immunoreactive bandwas abrogated (not shown).

Example 16 Detection of Mammary Associated Chemokine (MACK) in BreastCancer Sera Using Western Blotting

[0141] Aliquots (1.5 μl) of human sera were heated to 100° C. for 15 minin the presence of reducing agent (mercaptoethanol) and denaturant(sodium dodecyl sulfate (“SDS)) and were then subjected toSDS-polyacrylamide gel electrophoresis (“SDS-PAGE”) (as described inLaemmli, which is hereby incorporated by reference) in a 15% PAGE gel.After electrophoresis, the separated proteins were transferred to anitrocellulose membrane (0.2 μm pore) (Towbin et al., Proc. Natl. Acad.Sci. U.S.A., 76:4350-4354 (1979), which is hereby incorporated byreference). Non-specific protein binding sites on the membrane wereblocked with a solution containing bovine serum albumin (“BSA”) (2% intris-buffered saline, pH 7.4) for 1 hr. Thereafter, the membrane wasallowed to react for 1 hr with a {fraction (1/1000)} dilution ofpolyclonal (rabbit) antisera raised against synthetic peptidescorresponding to regions of the MACK gene product, as described inExamples 7 and 15. The membrane was washed thrice in tris-bufferedsaline and developed with avidin-biotin complex reagents (PierceChemicals) according to the recommendations of the manufacturer.Specific bands were revealed following the addition of insolublealkaline phosphatase substrate (BCIP/NBT).

[0142] The results, presented in Table 6, demonstrated the occurrence oftwo protein bands (one at 20-30 kDa and one at 7-12 kDa) specificallyfound in sera obtained from patients with breast cancer. Of 31 suchspecimens examined, 30 sera demonstrated both bands, while one specimen(number 1871) demonstrated the 20-30 kDa band only. In comparison, noneof 10 serum specimens obtained from patients with lymphoma or withprostatic, ovarian, lung, or colon cancers showed either of the specificbands when allowed to react with the antibodies to MACK. In addition,MACK peptide bands were not seen in sera obtained from 7 normalindividuals (Table 6). These results demonstrate that MACK orMACK-associated proteins are found in the circulation of individualswith cancer of the breast and that detection of these immunoreactivitiescan be of diagnostic and/or monitoring value for the disease. TABLE 6High Low Sample ID Diagnosis Stage Band¹ Band² 1008 Breast Cancerunknown + + 1869 Breast Cancer 3 + + 1870 Breast Cancer 3 + + 1871Breast Cancer 3 + − 1872 Breast Cancer 3 + + 1873 Breast Cancer 3 + +1874 Breast Cancer 3 + + 1875 Breast Cancer 3 + + 1876 Breast Cancer3 + + 1877 Breast Cancer 3 + + 1878 Breast Cancer 3 + + 1293 BreastCancer unknown + + 1294 Breast Cancer unknown + + 1296 Breast Cancerunknown + + 1297 Breast Cancer unknown + + 1298 Breast Cancerunknown + + 1299 Breast Cancer unknown + + 1300 Breast Cancerunknown + + 1301 Breast Cancer unknown + + 1302 Breast Cancerunknown + + 1303 Breast Cancer unknown + + 2694 Breast Cancer 2 + + 2697Breast Cancer 2 + + 2698 Breast Cancer 2 + + 4681 Breast Cancer 2 + +4682 Breast Cancer 2 + + 4683 Breast Cancer 2 + + 4684 Breast Cancer2 + + 4686 Breast Cancer 2 + + 4687 Breast Cancer 2 + + 4688 BreastCancer 2 + + 258 Lung Cancer 3 − − 259 Lung Cancer 2 − − 469 Lymphomaunknown − − 470 Lymphoma unknown − − 2486 Prostate Cancer D − − 2488Prostate Cancer D − − 1939 Ovarian Cancer 4 − − 1940 Ovarian Cancer 4 −− 1554 Colon Cancer C2 − − 1574 Colon Cancer C2 − − 1001 Normal − − 1002Normal − − 1003 Normal − − 1004 Normal − − 1005 Normal − − 1006 Normal −− 1007 Normal − −

[0143] Although the invention has been described in detail for thepurpose of illustration, it is understood that such detail is solely forthat purpose and variations can be made by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1 35 1 127 PRT Homo sapiens UNSURE (70)..(70) Xaa at position 70 iseither Arg or Gly 1 Met Gln Gln Arg Gly Leu Ala Ile Val Ala Leu Ala ValCys Ala Ala 1 5 10 15 Leu His Ala Ser Glu Ala Ile Leu Pro Ile Ala SerSer Cys Cys Thr 20 25 30 Glu Val Ser His His Ile Ser Arg Arg Leu Leu GluArg Val Asn Met 35 40 45 Cys Arg Ile Gln Arg Ala Asp Gly Asp Cys Asp LeuAla Ala Val Ile 50 55 60 Leu His Val Lys Arg Xaa Arg Ile Cys Val Ser ProHis Asn His Thr 65 70 75 80 Val Lys Gln Trp Met Lys Val Gln Ala Ala XaaLys Asn Gly Lys Gly 85 90 95 Asn Val Cys His Arg Lys Lys His His Gly LysArg Asn Ser Asn Arg 100 105 110 Ala His Gln Gly Lys His Glu Thr Tyr GlyHis Lys Thr Pro Tyr 115 120 125 2 104 PRT Homo sapiens UNSURE (47)..(47)Xaa at position 47 is either Arg or Gly 2 Leu Pro Ile Ala Ser Ser CysCys Thr Glu Val Ser His His Ile Ser 1 5 10 15 Arg Arg Leu Leu Glu ArgVal Asn Met Cys Arg Ile Gln Arg Ala Asp 20 25 30 Gly Asp Cys Asp Leu AlaAla Val Ile Leu His Val Lys Arg Xaa Arg 35 40 45 Ile Cys Val Ser Pro HisAsn His Thr Val Lys Gln Trp Met Lys Val 50 55 60 Gln Ala Ala Xaa Lys AsnGly Lys Gly Asn Val Cys His Arg Lys Lys 65 70 75 80 His His Gly Lys ArgAsn Ser Asn Arg Ala His Gln Gly Lys His Glu 85 90 95 Thr Tyr Gly His LysThr Pro Tyr 100 3 18 PRT Homo sapiens 3 Thr Glu Val Ser His His Ile SerArg Arg Leu Leu Glu Arg Val Asn 1 5 10 15 Met Cys 4 16 PRT Homo sapiens4 Lys Asn Gly Lys Gly Asn Val Cys His Arg Lys Lys His His Gly Lys 1 5 1015 5 19 PRT Homo sapiens 5 Asn Ser Asn Arg Ala His Gln Gly Lys His GluThr Tyr Gly His Lys 1 5 10 15 Thr Pro Tyr 6 3117 DNA Homo sapiens unsure(1)..(3117) n at any position in the sequence represents a or g or c ort/u 6 aacatcctca cttgtgttgc tgtcagtgcc tgtanggcag gcaggaatgc agcagagagg60 actcgccatc gtggccttgg ctgtctgtgc ggccctacat gcctcagaag ccatacttcc 120cattgcctcc agctgttgca cggaggtttc acatcatatt tccagaaggc tcctggaaag 180agtgaatatg tgtcgcatcc agagagctga tggggattgt gacttggctg ctgtcatcct 240tcatgtcaag cgcngaagaa tctgtgtcag cccgcacaac catactgtta agcagtggat 300gaaagtgcaa gctgccaana aaaatggtaa aggaaatgtt tgccacagga agaaacacca 360tggcaagagg aacagtaaca gggcacatca ggggaaacac gaaacatacg gccataaaac 420tccttattag agaatctaca gataaatcta cagagacaat cccccaagtg gacttggcca 480tgattggttg taagtttatc atctgaattc tccttattgt agacaacaga acaaaacaaa 540atattggttt ttaaaaaatg aacaattgtg ccgtatgcaa atgtacccaa taatatactc 600cactggaaaa tgaaatgaaa aaannatact ggctgggtat ggtgggtccc cccttttatc 660ccannnnctt cgggaggcag aggcaggagg atcacttgag accaggantt ngagacnagc 720tnggggcaaa anagcaanga cntcatttnt acaaacnaaa aaaaannttg gcccggcntg 780gtagnacttg cntataatcc cagcnacatg ggaggtngag gtgggaggat cacttgagtc 840tgggngagtt ngaggtngca gtgagcagcn tgggtgacag aatgnagacc ntgtctctaa 900aaataataat aataatgata gtgtatatct tcatataata ttttaagnag gagcatatag 960atataacttn ctcccaactt tttaattata gttttccaaa cttacagaga agttaaaaga 1020atggtacaat gaacatctat atatctttca ccacaatatt aatcattgtt aatattgtgc 1080cacatttgct ttctctctcc tctcttggta ggggttncaa tataaaatat tataactttt 1140aaaatatatc ttgttttgct aaccattgga aaataagttg caaaaatcat gacacttcac 1200ccctagtttc ttttnggtgt tataacttga cataccctaa aataaagaca tttttctaca 1260taatcacctt atcagtttta tacctaaaaa attaataatt tcatctaata tattccatat 1320tcaaattttc ccaactattt agagagcatt ttatgtagtt tttttttcac tccagtaatc 1380aatcaaggtn gacatacata ttgcaaataa ttgttatttt tctttaatat ctttcaatct 1440aagaaagttc ctctgtcttt tttttttaat ttttaaaatt attttgttga gggagggtct 1500tgctgtgtct tccaggctgg agtgcagtgg cacaattttg attttggctc actgaagcct 1560caactttagg gctcaagcaa tcctcccacc tcagcctncc cgagtatctg ggatcaaggt 1620gcatacccac cacacctggc taattttgtt tattttttgt agagacaggg tctcactatg 1680ttgcccaggt tgatctcaaa ctcctgggct caagcgatcc tcccacctta gcctcccaaa 1740gtactgggat tataggtgtg agccacagtg cctggcctaa ttattttctt gtgatcaaat 1800tcaggtttaa tgtttttggt taagaatttc ctacgtgaat tcgtgtactt attttgtcat 1860ttagagttca taaatattag ggtttatttt ctaaatagaa tagtttaaac taaatataac 1920ttcaaaacgt ctagtttgag tagctaccgt tgtttggatt gaaattttct gatactgaaa 1980agaacaaaaa gcctgccttt ctgcccanaa csnnttgcyt cccccagtna gttcttggng 2040cagnactagt tagggnccca gagttnggcc ttnngkgtgg tgattttang ytctgcctaa 2100acaaggngcn wacatytttt agctcctatt ccaccyttct namamgtttt tgttgtkgtt 2160tgnttgtttt tttkgagaca grrtntnayt ctgtttgccc argctggart tgcagtggca 2220caatytnggy tncattgcaa cytcngcytc cssgccgttc aaktgatyyt cttgcytcag 2280cytccccaag taantgatat tacaggngcc cagccaccam accccgntga wttttgtatt 2340tttartarar amrgggtttt cccgcnttgg cngggctggt ctcnaantcc ttgamctcna 2400ktgaaccacc cgcctgtgcc ycccaaantg ctggaattac cancgttgan ccaccatgcc 2460gggcycacac gtttgarttt ganaccattg tnccattcct cttttggcct yttttttntc 2520catagnngct tcaagataga tangtaagrg cccagtagtn gttcwtarga agcnmatagr 2580rancrggarc cantttnatc aggtgggcag gtgtccnngg cytccctgct ggytnntccc 2640aagcggtggt gttgccarga nktnttggar gtgataatgg gananaccag naggcmctga 2700gtyncnntag gttnaaatgc caccaaaact ggcctttggc ctaatatccy ycnttgamta 2760nttarcattt awtttattwa tttncctgac atttntgcma ncctttgtwt ttntatttcc 2820nctntatara wgargaaatt tgaggntytt araggtaaaa tganttgcnc nrgtnnacmc 2880aggaagtggc nraranaanc tttttanatn mgaaaaaatt aataaaatat aatatgagag 2940taacttaaaa tattaataaa ccacaatttt aaattaatta accgtgataa ccaacattaa 3000taaaagttaa gataccaaaa cactggtgtn taattttttn aactaacaan ttgaattatt 3060ttccatttta aattaattaa ccgtgataac caacattaat aaaagttaag ataccgn 3117 7381 NA Homo sapiens unsure (208)..(208) n may represent a or g or c ort/u 7 atgcagcaga gaggactcgc catcgtggcc ttggctgtct gtgcggccct acatgcctca60 gaagccatac ttcccattgc ctccagctgt tgcacggagg tttcacatca tatttccaga 120aggctcctgg aaagagtgaa tatgtgtcgc atccagagag ctgatgggga ttgtgacttg 180gctgctgtca tccttcatgt caagcgcnga agaatctgtg tcagcccgca caaccatact 240gttaagcagt ggatgaaagt gcaagctgcc aanaaaaatg gtaaaggaaa tgtttgccac 300aggaagaaac accatggcaa gaggaacagt aacagggcac atcaggggaa acacgaaaca 360tacggccata aaactcctta t 381 8 104 DNA Homo sapiens 8 acacgaattcacgtaggaaa ttcttaacca aaaacattaa acctgaattt gatcacaaga 60 aaataattaggccaggcact gtggctcaca cctataatcc cagt 104 9 25 DNA Homo sapiens 9gaattcacgt aggaaattct taacc 25 10 22 DNA Homo sapiens 10 actgggattataggtgtgag cc 22 11 311 DNA Homo sapiens unsure (101)..(101) n may be aor g or c or t/u 11 ggagagagcc gtatgtttcg tgtttcccct gatgtgccctgttactgttc ctcttgccat 60 ggtgtttctt cctgtggcaa acatttcctt taccatttttnttggcagct tgcactttca 120 tccactgctt aacagtatgg ttgtgcgggc tgacacagattnttctgcgc ttgacatgaa 180 ggatgacagc agccaagtca caatccccat cagctctctggatgcgacac atattcactc 240 tttccaggag ccttctggaa atatgatgtg aaacctccgtgcaacagctg gaggcaatgg 300 gaagtatggc t 311 12 20 DNA Artificial sequenceSequencing primer T7 12 taatacgact cactataggg 20 13 18 DNA Artificialsequence pCR3.1 Reverse Primer 13 tagaaggcac agtcgagg 18 14 22 DNAArtificial sequence Gene specific primer (24R) 14 actgggatta taggtgtgagcc 22 15 24 DNA Artificial sequence Gene specific primer (24R2) 15caaattcagg tttaatgttt ttgg 24 16 21 DNA Artificial sequence Genespecific primer (F4 ) 16 ctcaaacgtg tgagcccggc a 21 17 25 DNA Artificialsequence Gene specific primer (F3) 17 gctactcaaa ctagacgttt tgaag 25 1824 DNA Artificial sequence primers F8 18 ccgtatgttt cgtgtttccc ctga 2419 24 DNA Artificial sequence Primer R5 19 agccatactt cccattgcct ccag 2420 150 PRT Homo sapiens 20 Met Asn Leu Trp Leu Leu Ala Cys Leu Val AlaGly Phe Leu Gly Ala 1 5 10 15 Trp Ala Pro Ala Val His Thr Gln Gly ValPhe Glu Asp Cys Cys Leu 20 25 30 Ala Tyr His Tyr Pro Ile Gly Trp Ala ValLeu Arg Arg Ala Trp Thr 35 40 45 Tyr Arg Ile Gln Glu Val Ser Gly Ser CysAsn Leu Pro Ala Ala Ile 50 55 60 Phe Tyr Leu Pro Lys Arg His Arg Lys ValCys Gly Asn Pro Lys Ser 65 70 75 80 Arg Glu Val Gln Arg Ala Met Lys LeuLeu Asp Ala Arg Asn Lys Val 85 90 95 Phe Ala Lys Leu His His Asn Met GlnThr Phe Gln Ala Gly Pro His 100 105 110 Ala Val Lys Lys Leu Ser Ser GlyAsn Ser Lys Leu Ser Ser Ser Lys 115 120 125 Phe Ser Asn Pro Ile Ser SerSer Lys Arg Asn Val Ser Leu Leu Ile 130 135 140 Ser Ala Asn Ser Gly Leu145 150 21 95 PRT Homo sapiens 21 Met Cys Cys Thr Lys Ser Leu Leu LeuAla Ala Leu Met Ser Val Leu 1 5 10 15 Leu Leu His Leu Cys Gly Glu SerGlu Ala Ser Asn Phe Asp Cys Cys 20 25 30 Leu Gly Tyr Thr Asp Arg Ile LeuHis Pro Lys Phe Ile Val Gly Phe 35 40 45 Thr Arg Gln Leu Ala Asn Glu GlyCys Asp Ile Asn Ala Ile Ile Phe 50 55 60 His Thr Lys Lys Lys Leu Ser ValCys Ala Asn Pro Lys Gln Thr Trp 65 70 75 80 Val Lys Tyr Ile Val Arg LeuLeu Ser Lys Lys Val Lys Asn Met 85 90 95 22 94 PRT Homo sapiens 22 MetAla Pro Leu Lys Met Leu Ala Leu Val Thr Leu Leu Leu Gly Ala 1 5 10 15Ser Leu Gln His Ile His Ala Ala Arg Gly Thr Asn Val Gly Arg Glu 20 25 30Cys Cys Leu Glu Tyr Phe Lys Gly Ala Ile Pro Leu Arg Lys Leu Lys 35 40 45Thr Trp Tyr Gln Thr Ser Glu Asp Cys Ser Arg Asp Ala Ile Val Phe 50 55 60Val Thr Val Gln Gly Arg Ala Ile Cys Ser Asp Pro Asn Asn Gln Arg 65 70 7580 Val Lys Asn Ala Val Lys Tyr Leu Gln Ser Leu Glu Arg Ser 85 90 23 96PRT Homo sapiens 23 Met Gln Ile Ile Thr Thr Ala Leu Val Cys Leu Leu LeuAla Gly Met 1 5 10 15 Trp Pro Glu Asp Val Asp Ser Lys Ser Met Gln ValPro Phe Ser Arg 20 25 30 Cys Cys Phe Ser Phe Ala Glu Gln Glu Ile Pro LeuArg Ala Ile Leu 35 40 45 Cys Tyr Arg Asn Thr Ser Ser Ile Cys Ser Asn GluGly Leu Ile Phe 50 55 60 Lys Leu Lys Arg Gly Lys Glu Ala Cys Ala Leu AspThr Val Gly Trp 65 70 75 80 Val Gln Arg His Arg Lys Met Leu Arg His CysPro Ser Lys Arg Lys 85 90 95 24 77 PRT Homo sapiens 24 Ala Gln Pro AspSer Val Ser Ile Pro Ile Thr Cys Cys Phe Asn Val 1 5 10 15 Ile Asn ArgLys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile 20 25 30 Thr Asn IleGln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg 35 40 45 Gly Lys GluVal Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser 50 55 60 Met Lys HisLeu Asp Gln Ile Phe Gln Asn Leu Lys Pro 65 70 75 25 98 PRT Homo sapiens25 Met Lys Val Ser Ala Val Leu Leu Cys Leu Leu Leu Met Thr Ala Ala 1 510 15 Phe Asn Pro Gln Gly Leu Ala Gln Pro Asp Ala Leu Asn Val Pro Ser 2025 30 Thr Cys Cys Phe Thr Phe Ser Ser Lys Lys Ile Ser Leu Gln Arg Leu 3540 45 Lys Ser Tyr Val Ile Thr Thr Ser Arg Cys Pro Gln Lys Ala Val Ile 5055 60 Phe Arg Thr Lys Leu Gly Lys Glu Ile Cys Ala Asp Pro Lys Glu Lys 6570 75 80 Trp Val Gln Asn Tyr Met Lys His Leu Gly Arg Lys Ala His Thr Leu85 90 95 Lys Thr 26 97 PRT Homo sapiens 26 Met Lys Val Ser Ala Ala LeuLeu Trp Leu Leu Leu Ile Ala Ala Ala 1 5 10 15 Phe Ser Pro Gln Gly LeuAla Gly Pro Ala Ser Val Pro Thr Thr Cys 20 25 30 Cys Phe Asn Leu Ala AsnArg Lys Ile Pro Leu Gln Arg Leu Glu Ser 35 40 45 Tyr Arg Arg Ile Thr SerGly Lys Cys Pro Gln Lys Ala Val Ile Phe 50 55 60 Lys Thr Lys Leu Ala LysAsp Ile Cys Ala Asp Pro Lys Lys Lys Trp 65 70 75 80 Val Gln Asp Ser MetLys Tyr Leu Asp Gln Lys Ser Pro Thr Pro Lys 85 90 95 Pro 27 99 PRT Homosapiens 27 Met Lys Ala Ser Ala Ala Leu Leu Cys Leu Leu Leu Thr Ala AlaAla 1 5 10 15 Phe Ser Pro Gln Gly Leu Ala Gln Pro Val Gly Ile Asn ThrSer Thr 20 25 30 Thr Cys Cys Tyr Arg Phe Ile Asn Lys Lys Ile Pro Lys GlnArg Leu 35 40 45 Glu Ser Tyr Arg Arg Thr Thr Ser Ser His Cys Pro Arg GluAla Val 50 55 60 Ile Phe Lys Thr Lys Leu Asp Lys Glu Asp Cys Ala Asp ProThr Gln 65 70 75 80 Lys Trp Val Gln Asp Pro Met Lys His Leu Asp Lys LysThr Gln Thr 85 90 95 Pro Lys Leu 28 99 PRT Homo sapiens 28 Met Lys ValSer Ala Ala Leu Leu Cys Leu Leu Leu Thr Ala Ala Ala 1 5 10 15 Phe IlePro Gln Gly Leu Ala Gln Pro Asp Ala Ile Asn Ala Pro Val 20 25 30 Thr CysCys Tyr Asn Phe Thr Asn Arg Lys Ile Ser Val Gln Arg Leu 35 40 45 Ala SerTyr Arg Arg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val 50 55 60 Ile PheLys Thr Ile Val Ala Lys Glu Asp Cys Ala Asp Pro Lys Gln 65 70 75 80 LysTrp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr 85 90 95 ProLys Thr 29 91 PRT Homo sapiens 29 Met Lys Val Ser Ala Ala Arg Leu AlaVal Ile Leu Ile Ala Thr Ala 1 5 10 15 Leu Cys Ala Pro Ala Ser Ala SerPro Tyr Ser Ser Asp Thr Thr Pro 20 25 30 Cys Cys Phe Ala Tyr Ile Ala ArgPro Leu Pro Arg Ala His Ile Lys 35 40 45 Glu Tyr Phe Tyr Thr Ser Gly LysCys Ser Asn Pro Ala Val Val Phe 50 55 60 Val Thr Arg Lys Asn Arg Gln ValCys Ala Asn Pro Glu Lys Lys Trp 65 70 75 80 Val Arg Glu Tyr Ile Asn SerLeu Glu Met Ser 85 90 30 93 PRT Homo sapiens 30 Met Lys Ile Ser Val AlaAla Ile Pro Phe Phe Leu Leu Ile Thr Ile 1 5 10 15 Ala Leu Gly Thr LysThr Glu Ser Ser Ser Arg Gly Pro Tyr His Pro 20 25 30 Ser Glu Cys Cys PheThr Tyr Thr Thr Tyr Lys Ile Pro Arg Gln Arg 35 40 45 Ile Met Asp Tyr TyrGlu Thr Asn Ser Gln Cys Ser Lys Pro Gly Ile 50 55 60 Val Phe Ile Thr LysArg Gly His Ser Val Cys Thr Asn Pro Ser Asp 65 70 75 80 Lys Trp Val GlnAsp Tyr Ile Lys Asp Met Lys Glu Asn 85 90 31 92 PRT Homo sapiens 31 MetLys Leu Cys Val Thr Val Leu Ser Leu Leu Met Leu Val Ala Ala 1 5 10 15Phe Cys Ser Pro Ala Leu Ser Ala Pro Met Gly Ser Asp Pro Pro Thr 20 25 30Ala Cys Cys Phe Ser Tyr Thr Ala Arg Lys Leu Pro Arg Asn Phe Val 35 40 45Val Asp Tyr Tyr Glu Thr Ser Ser Leu Cys Ser Gln Pro Ala Val Val 50 55 60Phe Gln Thr Lys Arg Ser Lys Gln Val Cys Ala Asp Pro Ser Glu Ser 65 70 7580 Trp Val Gln Glu Tyr Val Tyr Asp Leu Glu Leu Asn 85 90 32 93 PRT Homosapiens 32 Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr MetAla 1 5 10 15 Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp ThrPro Thr 20 25 30 Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln AsnPhe Ile 35 40 45 Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro SerVal Ile 50 55 60 Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro SerGlu Glu 65 70 75 80 Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala85 90 33 92 PRT Homo sapiens 33 Met Gln Val Ser Thr Ala Ala Leu Ala ValLeu Leu Cys Thr Met Ala 1 5 10 15 Leu Cys Asn Gln Phe Ser Ala Ser LeuAla Ala Asp Thr Pro Thr Ala 20 25 30 Cys Cys Phe Ser Tyr Thr Ser Arg GlnIle Pro Gln Asn Phe Ile Ala 35 40 45 Asp Tyr Phe Glu Thr Ser Ser Gln CysSer Lys Pro Gly Val Ile Phe 50 55 60 Leu Thr Lys Arg Ser Arg Gln Val CysAla Asp Pro Ser Glu Glu Trp 65 70 75 80 Val Gln Lys Tyr Val Ser Asp LeuGlu Leu Ser Ala 85 90 34 89 PRT Homo sapiens 34 Met Lys Gly Leu Ala AlaAla Leu Leu Val Leu Val Cys Thr Met Ala 1 5 10 15 Leu Cys Ser Cys AlaGln Val Gly Thr Asn Lys Glu Leu Cys Cys Leu 20 25 30 Val Tyr Thr Ser TrpGln Ile Pro Gln Lys Phe Ile Val Asp Tyr Ser 35 40 45 Glu Thr Ser Pro GlnCys Pro Lys Pro Gly Val Ile Leu Leu Thr Lys 50 55 60 Arg Gly Arg Gln AspCys Ala Asp Pro Asn Lys Lys Trp Val Gln Lys 65 70 75 80 Tyr Ile Ser AspLeu Lys Leu Asn Ala 85 35 104 DNA Homo sapiens 35 acacgaattc acgtaggaaattcttaacca aaaacattaa acctgaattt gatcacaaga 60 aaataattag gccaggcactgtggctcaca cctataatcc cagt 104

What is claimed:
 1. A method for treating breast disease in a patient inneed thereof, which method comprises administering to the patient aneffective amount of a chemokine, which chemokine has about 105 to about127 amino acids, has a deduced molecular weight of from about 12 toabout 14 kD, has a deduced isoionic point of from about pH 10.1 to aboutpH 10.7, and comprises at least one amino acid sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5;wherein the breast disease is selected from the group consisting ofbenign cystitis, benign hyperplasia, cancer and malignancies.
 2. Themethod according to claim 1, wherein the chemokine has an amino acidsequence comprising each of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.3. The method according to claim 2, wherein the chemokine has an aminoacid sequence as depicted in SEQ ID NO:1.
 4. The method according toclaim 1, wherein the peptide is administered orally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally,intraocularly, intraarterially, by intranasal instillation, byintracavitary instillation, by intravesical instillation, or byapplication to mucous membranes.
 5. A composition comprising achemokine, wherein the chemokine has about 105 to about 127 amino acids,has a deduced molecular weight of from about 12 to about 14 kD, has adeduced isoionic point of from about pH 10.1 to about pH 10.7, andcomprises at least one an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein thepeptide is present in the composition in an amount effective to treat abreast disease.
 6. The composition according to claim 5, farthercomprising at least one component selected from the group consisting ofcarriers, excipients, diluents, binders, disintegrating agents,lubricants, adjuvants, surfactants, propellants, and stabilizers.
 7. Adosage unit form comprising the composition according to claim
 6. 8. Thedosage unit form according to claim 7, selected from the groupconsisting of tablets, capsules, powders, solutions, suspensions,aerosols, and emulsions.
 9. A method for treating breast disease in apatient in need thereof, which method comprises administering the dosageunit form according to claim
 9. 10. A method for treating breast diseasein a patient in need thereof, which method comprises administering tothe patient an effective amount of an antibody or a binding portionthereof which recognizes a chemokine, wherein the chemokine has about105 to about 127 amino acids, has a deduced molecular weight of fromabout 12 to about 14 kD, has a deduced isoionic point of from about pH10.1 to about pH 10.7, and comprises at least one an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQID NO:5; wherein the breast disease is selected from the groupconsisting of inflammation, infection, and mastitis.
 11. The methodaccording to claim 10, wherein the chemokine has an amino acid sequencecomprising each of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
 12. Themethod according to claim 11, wherein the chemokine has an amino acidsequence as depicted in SEQ ID NO:1.
 13. The method according to claim10, wherein the antibody or binding portion thereof is administeredorally, parenterally, subcutaneously, intravenously, intramuscularly,intraperitoneally, intraocularly, intraarterially, by intranasalinstillation, by intracavitary instillation, by intravesicalinstillation, or by application to mucous membranes.
 14. A compositioncomprising an antibody or a binding portion thereof which recognizes achemokine, wherein the chemokine has about 105 to about 127 amino acids,has a deduced molecular weight of from about 12 to about 14 kD, has adeduced isoionic point of from about pH 10.1 to about pH 10.7, andcomprises at least one an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein theantibody or binding portion thereof is present in the composition in anamount effective to treat a breast disease.
 15. The compositionaccording to claim 14, further comprising at least one componentselected from the group consisting of biological agents, carriers,excipients, diluents, binders, disintegrating agents, lubricants,adjuvants, surfactants, propellants, and stabilizers.
 16. A dosage unitform comprising the composition according to claim
 14. 17. The dosageunit form according to claim 16, selected from the group consisting oftablets, capsules, powders, solutions, suspensions, aerosols, andemulsions.
 18. A method for treating breast disease in a patient in needthereof, which method comprises administering the dosage unit formaccording to claim
 17. 19. The method according to claim 11, wherein theantibody is conjugated to a cytotoxic drug.
 20. The method according toclaim 19, wherein the cytotoxic drug is selected from the groupconsisting of a therapeutic drug; a radioactive compound; a molecule ofplant, fungal, or bacterial origin; a biological protein; and mixturesthereof.
 21. The method according to claim 12, wherein the method isused in conjunction with surgery, radiation, crysosurgery,thermotherapy, hormone treatment, chemotherapy, and vaccines.
 22. Amethod for vaccinating against a breast disease in a patient in needthereof, which method comprises administering to the patient aneffective amount of an antigenic portion of a chemokine with aneffective amount of an adjuvant, wherein the chemokine has about 105 toabout 127 amino acids, has a deduced molecular weight of from about 12to about 14 kD, has a deduced isoionic point of from about pH 10.1 toabout pH 10.7, and comprises at least one amino acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5;wherein the breast disease is selected from the group consisting ofinflammation, infection, and mastitis.
 23. A composition comprising (i)an antigenic portion of a chemokine, wherein the chemokine has about 105to about 127 amino acids, has a deduced molecular weight of from about12 to about 14 kD, has a deduced isoionic point of from about pH 10.1 toabout pH 10.7, and comprises at least one an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQID NO:5, wherein the antigenic portion of a chemokine is present in thecomposition in an amount effective to vaccinate against a breastdisease; and (ii) an adjuvant.
 24. A dosage unit form comprising thecomposition according to claim
 23. 25. The dosage unit form according toclaim 24, selected from the group consisting of tablets, capsules,powders, solutions, suspensions, aerosols, and emulsions.
 26. A methodfor vaccinating against a breast disease in a patient in need thereof,which method comprises administering the dosage unit form according toclaim 25.